JP2016175897A - Monoclonal antibodies against rgm a protein, and uses thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide compositions useful for detecting RGM A and for inhibiting RGM A activity, in a human suffered from diseases including multiple sclerosis, mammalian brain injury, spinal cord injury, apoplexy, neurodegenerative disease, and schizophrenia.SOLUTION: The invention relates to a production of isolated proteins, particularly monoclonal antibodies that bind to and neutralize RGM A proteins. Specifically, the antibodies have the ability to interfere the interaction of RGM A into the receptor and/or coreceptor of RGM A, i.e., neogenin, and BMP-2, BMP-4, and to inhibit binding.SELECTED DRAWING: None

Description

(Technical field)
The present application relates to RGM A binding proteins, particularly those that have the ability to bind RGM A, interfere with the binding of RGM protein to RGM A receptors and other RGM A binding proteins, and thus neutralize the function of RGM A. Monoclonal antibodies and in particular their CDR grafts, humanized forms are described. These antibodies may have utility in the treatment of several situations including, but not limited to, multiple sclerosis, mammalian brain trauma, spinal cord injury, stroke, neurodegenerative diseases and schizophrenia.

(Background information)
Axonal regeneration within the mammalian central nervous system (CNS) after injury or after an inflammatory challenge or after a neurodegenerative disease is almost impossible and the outcome is the intrinsic nature of nerve fibers in the CNS. Depending on the balance between the ability to regrow and the inhibitory factor in the CNS that is localized in the microenvironment of the lesion or injury site, this inhibitory factor causes regrowth and thus regeneration of damaged fiber tracts. Actively obstruct.

  CNS myelin produced by oligodendrocytes and lesional scars is an axis in the early stages of injury because it causes growth cone collapse and neurite outgrowth inhibition in vitro and in vivo, thereby directly inhibiting axonal regrowth. It has proven to be the most important unacceptable structure for cord growth. RGM protein, a major inhibitory factor in CNS myelin and scar tissue, has been identified (Monnier et al., Nature 419: 392-395, 2002; Schwab et al., Arch. Neurol. 62: 1561-8, 2005a; Schwab et al. Eur. J. Neurosci. 21: 1569-76, 2005b; Hata et al., J. Cell. Biol. 173: 47-58, 2006; for reviews: Mueller et al., Philos. Trans. R. Soc. Sci. 361: 1513-29, 2006; see Yamashita et al., Curr. Opin. Neurobiol. 17: 29-34, 2007). RGM protein is up-regulated at the site of injury or lesions in humans who died from brain trauma or ischemic injury (Schwab et al., Arch. Neurol. 62: 1561-8, 2005a) and up-regulated at lesion sites in rats with spinal cord injury (Schwab et al., Eur. J. Neurosci. 21: 1569-76, 2005b; Hata et al., J. Cell. Biol. 173: 47-58, 2006, for reviews: Mueller et al., Philos. Trans. R. Soc.Lond.B Biol.Sci.361: 1513-29, 2006; Yamashita et al., Curr.Opin.Neurobiol.17: 29-34, 2007). Furthermore, initial data using clinical samples from multiple sclerosis patients and healthy individuals suggests that human RGM A is upregulated in the cerebrospinal fluid of patients suffering from MS (data Is not shown.)

  In order to evaluate the ability of the RGM A specific polyclonal antibody to promote regeneration, the antibody was administered to a moderate to severe model of spinal cord injury in which approximately 60% of the spinal cord at the 9/10 thoracic vertebrae was transected. Histological examination revealed that such damage cut all dorsolateral fibers of the corticospinal tract. RGM A-specific polyclonal antibody administered locally via a pump for 2 weeks induced long-range regeneration of damaged nerve fibers (Hata et al., J. Cell. Biol. 173: 47 -58, 2006).

  Hundreds of nerve fibers extended beyond the lesion site, and the longest fibers regenerated more than 10 mm beyond the lesion, whereas control antibody treated animals showed no regenerated fibers distal to the lesion. Compared to spinal cord injury rats treated with control antibody, the functional recovery of anti-RGM A treated rats is significantly improved, so that RGM A is a potent nerve regeneration inhibitor and is characterized by axonal or nerve fiber damage (Hata et al., J. Cell. Biol. 173: 47-58, 2006; Kyoto et al., Brain Res. 1186: 74-86, 2007). Furthermore, neutralizing RGM A protein with a polyclonal antibody that blocks function not only stimulates regrowth of damaged nerve fibers in spinal cord injured rats, but also promotes their synaptogenesis, thereby causing damage Can be reshaped or restored (Kyoto et al., Brain Res. 1186: 74-86, 2007).

  The rgm gene family includes three different genes, two of which, rgma a and b, are expressed in the mammalian CNS from which the RGM A and RGM B proteins are derived, while being the third member rgmc is expressed peripherally (Mueller et al., Philos. Trans. R. Soc. London. B Biol. Sci. 361: 1513-29, 2006), where RGM C plays an important role in iron metabolism . RGM A inhibits neurite outgrowth by binding to neogenin, which has been identified as an RGM receptor, in vitro (Rajagopalan et al., Nat Cell Biol .: 6 (8), 756-62, 2004). Neogenin was first described as a netrin-binding protein (Keino-Masu et al., Cell, 87 (2): 175-85, 1996). This is an important finding because binding of netrin-1 to neogenin or its related receptor DCC (deficient in colorectal cancer) has been reported to stimulate rather than inhibit neurite outgrowth. (Braisted et al., J. Neurosci. 20: 5792-801, 2000). Therefore, by blocking RGM A, allowing neogenin to bind to its neurite growth stimulating ligand, netrin, eliminates RGM-mediated growth inhibition. Based on these observations, neutralization of RGM A can be evaluated as superior to neutralization of neogenin in a human spinal cord injury model. In addition to RGM A binding to neogenin and induction of neurite outgrowth inhibition, binding of RGM A or B to bone morphogenetic proteins BMP-2 and BMP-4 is similar to successful nerve regeneration and functional recovery. It can be an obstacle (Mueller et al., Philos. Trans. R. Soc. London. Biol. Sci. 361: 1513-29, 2006).

Monnier et al., Nature 419, 2002, p. 392-395 Schwab et al., Arch. Neurol. 62, 2005, p. 1561-8, Schwab et al., Eur. J. et al. Neurosci. 21, 2005, p. 1569-76 Hata et al. Cell. Biol. 173, 2006, p. 47-58 Mueller et al., Philos. Trans. R. Soc. London. B Biol. Sci. 361, 2006, p. 1513-29 Yamashita et al., Curr. Opin. Neurobiol. 17, 2007, p. 29-34 Kyoto et al., Brain Res. 1186, 2007, p. 74-86 Rajagopalan et al., Nat Cell Biol. : 6 (8), 2004, p. 756-62 Keino-Masu et al., Cell, 87 (2), 1996, p. 175-85 Braisted et al. Neurosci. 20, 2000, p. 5792-801

  In the art, an improved antibody capable of binding to RGM A, preferably blocking RGM A, between RGM A and its receptor and / or binding proteins, namely neogenin and BMP-2, BMP-4. There is a need in the art for monoclonal antibodies that interfere with the interaction.

  The application relates to (a) a neutralizing monoclonal antibody against RGM A that selectively inhibits the binding of RGM A to the receptor neogenin of RGM A and to bone morphogenetic proteins 2 and 4 (BMP-2, BMP-4). Production and (b) Generation of neutralizing monoclonal antibodies against RGM A that selectively inhibits binding of RGM A to bone morphogenetic proteins 2 and 4 (BMP-2, BMP-4). Under conditions where neurons are prone to migrate and elongate on RGM A substrates and not on permissive substrates such as collagen I, one of the neutralizing monoclonal antibodies of the present invention is the inhibitory property of RGM A. The neutralizing monoclonal antibodies of the present invention are expected to stimulate the regrowth of damaged or damaged nerve fibers and the formation of functional synapses of regenerating nerve fibers. Furthermore, this antibody can induce long-range regeneration in an in vivo rat model of optic nerve injury, which also promotes remyelination of nerve fibers that have been damaged and regenerated.

  Therefore, the neutralizing monoclonal antibody of the present invention is used in damaged and inflamed human CNS, for example, in multiple sclerosis, acute spinal cord injury, after brain trauma, or for example, Huntington's chorea, Parkinson's disease, Alzheimer's disease, etc. It is expected to promote nerve regeneration and regrowth of damaged or broken neuronal connections in other neurodegenerative diseases.

(Summary of the Invention)
According to one aspect, the present invention provides a binding protein that dissociates from human RGM A with 1x10 ^-7 M following _{K D} and ^1x10 -2 ^{s -1} following _{k off} rate constant (measured by both are surface plasmon resonance) To do.

  According to another aspect, the present invention binds to human RGM A and, for example, as described above when measured in a standard in vitro assay such as the Ntera nerve extension assay as illustrated in Example 3 below. It relates to binding proteins that neutralize the neurite outgrowth inhibitory activity of human RGM A, such as binding proteins that exhibit kinetic properties.

The present invention also provides at least one of the following additional functional properties:
Binding to rat RGM A,
It also relates to a binding protein as defined above having binding to human RGM C and binding to rat RGM C.

  In particular, a binding protein as described herein modulates the ability of RGM to bind to at least one of its receptors.

  Such binding proteins specifically bind to the receptor binding domain of human RGM A. For RGM A, N and C-terminal receptor binding domains have been identified. Particular embodiments of the binding proteins of the invention are illustrated by the inhibition of binding between an N-terminal hRGM A fragment such as 47-168 and a receptor molecule such as neogenin and BMP-4, for example, It binds to the N-terminal receptor binding domain of RGM A. The full length of the N-terminal hRGM A fragment can be about 30 to about 150 or about 30 to about 122 amino acid residues. Non-limiting examples may include fragment 0 of hRGM A (corresponding to N-terminal residues 47-168) or any shorter receptor binding fragment as described herein.

In particular, the binding protein has at least one of the following interactions:
Binding of human RGM A to human BMP-4,
Binding of hRGM A to human neogenin,
Binding of hRGM C to human neogenin,
Binding of human RGM A to human BMP-2,
Is regulated, preferably inhibited.

  According to certain embodiments, the binding protein as defined herein is a humanized antibody.

A binding protein as described above can have an antigen binding domain, which can bind to an epitope of an RGM molecule, the antigen binding domain being
GTTPDY (SEQ ID NO: 59),
FQATHDPLT (SEQ ID NO: 62),
ARRNEYYGSSSFFDY (SEQ ID NO: 65),
LQGYIPPRT (SEQ ID NO: 68) and at least one CDR comprising an amino acid sequence selected from the group consisting of a modified CDR amino acid sequence having at least 50% sequence identity with one of said sequences. In another embodiment, the present invention provides a binding protein comprising an antigen binding domain (which can bind to an epitope of an RGM molecule, wherein the antigen binding domain is
GTTPDY (SEQ ID NO: 59),
FQATHDPLT (SEQ ID NO: 62),
ARRNEYYGSSSFFDY (SEQ ID NO: 65),
LQGYIPPRT (SEQ ID NO: 68) and modified CDR amino acids having at least 50% sequence identity with one of the sequences, such as at least 55, 60, 65, 70, 75, 80, 85, 90, 95% identity, etc. At least one CDR comprising an amino acid sequence selected from the group consisting of: )

  For example, the binding protein can comprise two of the CDRs, eg, SEQ ID NOs: 59 and 62; or SEQ ID NOs: 65 and 68; at least one of these CDRs can be modified and one of the sequences And at least 50% sequence identity, such as at least 55, 60, 65, 70, 75, 80, 85, 90, 95% identity.

  This binding protein has SEQ ID NOs: 57, 58, 60, 61, 63, 64, 66, 67 and at least 50% sequence identity with one of the sequences, eg at least 55, 60, 65, 70, 75, 80 , 85, 90, 95% identity, etc., and can further comprise at least one CDR comprising an amino acid sequence selected from the group consisting of modified CDR amino acid sequences.

  In another embodiment, the at least one CDR is

からなる群から選択されるアミノ酸配列を含む、結合タンパク質が提供される。

A binding protein comprising an amino acid sequence selected from the group consisting of is provided.

  In certain embodiments, the binding protein is

からなる可変ドメインＣＤＲセット
又は表２の３つのＣＤＲの少なくとも１つが、親配列に対して少なくとも５０％の配列同一性、例えば少なくとも５５、６０、６５、７０、７５、８０、８５、９０、９５％の同一性など、を有する修飾ＣＤＲアミノ酸配列である可変ドメインセット
から選択される、少なくとも３つのＣＤＲを含む。

Or at least one of the three CDRs of Table 2 has at least 50% sequence identity to the parent sequence, eg at least 55, 60, 65, 70, 75, 80, 85, 90, 95 It comprises at least three CDRs selected from a variable domain set that is a modified CDR amino acid sequence having, for example,% identity.

  In particular, each of the aforementioned modifications can be effected by one or more amino acid additions, deletions or in particular substitutions or combinations thereof.

  In another embodiment, the binding protein comprises at least two variable domain CDR sets.

In particular, the at least two variable domain CDR sets are
VH5F9 set and VL5F9 set; and VH8D1 set and VL8D1 set.

  The binding protein according to the invention further comprises a human acceptor framework.

  This human acceptor framework is a group consisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, and 33. At least one amino acid sequence selected from:

The binding proteins of the invention are in particular set:
(1)
VH3-48 set (SEQ ID NOS: 15, 16, and 17)
VH3-33 set (SEQ ID NOS: 21, 22, and 23)
VH3-23 set (SEQ ID NO: 24, 25 and 26) (each of these sets is
JH3 (SEQ ID NO: 18)
JH4 (SEQ ID NO: 19)
JH6 (SEQ ID NO: 20)
Combined with an additional framework sequence selected from )
Or (2) selected from the group consisting of
A18 set: (SEQ ID NO: 27, 28 and 29)
A17 set: (SEQ ID NO: 31, 32 and 33) (each of these sets is combined with an additional framework sequence selected from JK2 (SEQ ID NO: 2)).
At least one set of framework sequences selected from the group consisting of:

  According to a particular embodiment, the binding protein according to any one of the preceding claims is at least one CDR selected from SEQ ID NO: 35, 36, 37, 38, 39, 40, 41, 42 and 43. A graft heavy chain variable domain; and / or at least one CDR grafted light chain variable domain selected from SEQ ID NOs: 44, 45 and 46.

In particular, the binding protein of the present invention comprises a combination of two variable domains, the two variable domains being
SEQ ID NOs: 35 and 44; 36 and 44; 37 and 44; 38 and 44; 39 and 44; 40 and 44; 41 and 44; 42 and 44;
SEQ ID NOs: 35 and 45; 36 and 45; 37 and 45; 38 and 45; 39 and 45; 40 and 45; 41 and 45; 42 and 45;
SEQ ID NOs: 35 and 46; 36 and 46; 37 and 46; 38 and 46; 39 and 46; 40 and 46; 41 and 46; 42 and 46;
Having an amino acid sequence selected from

In another embodiment of the invention, the human acceptor framework of the binding protein comprises at least one framework region amino acid substitution in a key residue, the key residue comprising:
Residues adjacent to the CDRs;
Glycosylation site residues;
Rare residue;
Residues capable of interacting with the RGM epitope;
Residues capable of interacting with CDRs;
Canonical residues;
Contact residues between the heavy and light chain variable regions;
Residues in the vernier zone;
A group consisting of an N-terminal residue capable of paraglutamate formation and a residue in a region overlapping between the variable heavy chain CDR1 defined by Chothia and the first heavy chain framework defined by Kabat Selected from.

In particular, this key residue is
(Heavy chain sequence position): 1, 5, 37, 48, 49, 88, 98
(Light chain sequence position): 2, 4, 41, 51
Selected from the group consisting of

  In certain embodiments, a binding protein of the invention is or comprises a consensus human variable domain.

  According to another embodiment of the binding protein of the invention, the human acceptor framework comprises at least one framework region amino acid substitution, the amino acid sequence of the framework comprising at least the sequence of the human acceptor framework, 65%, such as at least 70, 75, 80, 85, 90, 95, 96, 97, 98 or 99% identical and at least 70 amino acid residues, such as at least 75, identical to this human acceptor framework 80 or 85 residues.

According to a particular embodiment, the binding protein of the invention comprises
At least one amino acid sequence selected from the group consisting of SEQ ID NOs: 47, 48, 49, 50; (VH domain) and / or selected from the group consisting of SEQ ID NOs: 51, 52, 53 and 54 (VL domain) Contains one framework mutation variable domain.

In particular, the binding protein comprises two, possibly variable framework framework variable domains, the two variable domains:
SEQ ID NOs: 47 and 44; 47 and 45; 47 and 46; 47 and 51; 47 and 52; 47 and 53; 47 and 54;
48 and 44; 48 and 46; 48 and 51; 48 and 52; 48 and 53; 48 and 54;
49 and 44; 49 and 46; 49 and 51; 49 and 52; 49 and 53; 49 and 54;
SEQ ID NOs: 50 and 44; 50 and 45; 50 and 46; 50 and 51; 50 and 52; 50 and 53; 50 and 54
Having an amino acid sequence selected from the group consisting of

  The binding proteins of the invention as described herein are capable of binding to at least one target selected from RGM molecules.

  In particular, they may bind to human RGM A and optionally at least one further human-derived RGM molecule or those derived from cynomolgus monkey, rat, chick, frog and fish.

  For example, they may further bind to rat RGM A, human RGM C and / or rat RGM C.

  In particular, the binding proteins of the invention can modulate the biological function of a target selected from RGM molecules as defined above, in particular neutralize or inhibit.

  In particular, the binding proteins of the present invention modulate, particularly inhibit, the ability of RGM to bind to at least one of the RGM receptors, such as neogenin and BMP (such as BMP-2 and BMP-4).

For example, this binding protein has the following interaction:
Binding of human RGM A to human BMP-4,
Binding of hRGM A to human neogenin,
Binding of hRGM C to human neogenin,
Modulates, in particular suppresses and preferably inhibits at least one of the binding of human RGM A to human BMP-2.

  Also within the scope of the invention are binding proteins with various combinations of functional properties and as a result exhibiting various functional profiles as disclosed herein. Non-limiting examples of such profiles are listed below:

例えば、プロファイル１は、本発明により提供されるような抗体５Ｆ９及び本明細書中に記載のその誘導体に対応する。

For example, profile 1 corresponds to antibody 5F9 as provided by the present invention and its derivatives described herein.

  For example, profile 2 corresponds to antibody 8D1 as provided by the present invention and derivatives thereof as described herein.

  In particular, the binding proteins of the invention can inhibit at least one biological activity of RGM, in particular RGM A, which is selected from human, cynomolgus monkey, rat, chick, frog and fish.

According to another embodiment, the binding protein of the invention has the following kinetic properties:
(A) at least about 10 ² M ^-1 s ^-1 ; at least about 10 ³ M ^-1 s ^-1 ; at least about 10 ⁴ M ^-1 s ^-1 ; at least about 10 ⁵ M ^-1 s ^-1 ; at least about 10 An association rate constant (k _on ) for said target selected from the group consisting of ⁶ M ⁻¹ s ⁻¹ and at least about 10 ⁷ M ⁻¹ s ⁻¹ (as measured by surface plasmon resonance);
(B) up to about 10 ⁻² s ⁻¹ ; up to about 10 ⁻³ s ⁻¹ ; up to about 10 ⁻⁴ s ⁻¹ ; up to about 10 ⁻⁵ s ⁻¹ ; and up to about 10 ⁻⁶ s ⁻¹ (surface Dissociation rate constant (k _off ) for said target selected from the group consisting of (as measured by plasmon resonance) or (c) up to about 10 ⁻⁷ M; up to about 10 ⁻⁸ M; up to about 10 ⁻⁹ M A dissociation constant (K _D ) for said target selected from the group consisting of: up to about 10 ⁻¹⁰ M; up to about 10 ⁻¹¹ M; up to about 10 ⁻¹² M; and up to 10 ⁻¹³ M;
1 or more.

  According to a further aspect, the present invention provides an antibody construct comprising a binding protein as described above and further comprising a linker polypeptide or an immunoglobulin constant domain.

  The antibody construct or binding protein of the present invention is an immunoglobulin molecule, monoclonal antibody, chimeric antibody, CDR grafted antibody, humanized antibody, Fab, Fab ′, F (ab ′) 2, Fv, disulfide bond Fv, scFv, single It can be selected from the group consisting of domain antibodies, diabodies, multispecific antibodies, bispecific antibodies, bivariable domain immunoglobulins and bispecific antibodies.

In the antibody construct according to the invention, this binding protein is
A human IgM constant domain,
Human IgG1 constant domain,
Human IgG2 constant domain,
Human IgG3 constant domain,
Human IgG4 constant domain,
Human IgE constant domain,
A human IgD constant domain,
A human IgA1 constant domain,
A human IgA2 constant domain,
A heavy immunoglobulin constant domain selected from the group consisting of a human IgY constant domain and a corresponding mutated constant domain.

  In particular, the antibody construct according to the present invention comprises an immunoglobulin constant domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 12, 13, and 14.

  According to another aspect, the invention includes an antibody construct as described herein, wherein an immunoadhesion molecule, a contrast agent, a therapeutic agent and a cytotoxic agent (each of these agents bind to the binding protein). An antibody conjugate further comprising an agent selected from the group consisting of:

For example, the agent is a contrast agent selected from the group consisting of a radioactive label, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, and biotin. In particular, the contrast agent is a radioactive label selected from the group consisting of ³ H, ¹⁴ C, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹²⁵ I, ¹³¹ I, ¹⁷⁷ Lu, ¹⁶⁶ Ho, and ¹⁵³ Sm. .

  For example, the agent is a therapeutic agent selected from the group consisting of antimetabolites, alkylating agents, antibiotics, growth factors, cytokines, angiogenesis inhibitors, antimitotic agents, anthracyclines, toxins and apoptotic agents. Or a cytotoxic agent.

  According to another embodiment, a binding protein of the invention as described herein retains a human glycosylation pattern.

  Furthermore, the binding proteins, antibody constructs and antibody complexes according to the invention may preferably exist as crystals (in crystalline form) that retain biological activity.

  In particular, the crystals are carrier-free pharmaceutical controlled release crystals. In view of this crystalline form, the binding protein, antibody construct or antibody complex may have a longer half-life in vivo than the corresponding soluble form.

  In another aspect, the invention provides an isolated nucleic acid encoding a binding protein amino acid sequence, an antibody construct amino acid sequence, and an antibody complex amino acid sequence, as described herein.

  The invention also relates to a vector comprising the isolated nucleic acid as described herein. In particular, the vector is selected from the group consisting of pcDNA, pTT, pTT3, pEFBOS, pBV, pJV and pBJ.

  The invention also relates to host cells containing such vectors. In particular, the host cell is e.g. Prokaryotic cells such as E. coli or eukaryotic cells, which can be selected from the group consisting of protist cells, animal cells, plant cells and fungal cells. In particular, the eukaryotic cell is an animal cell selected from the group consisting of mammalian cells, avian cells and insect cells. Preferably, the host cell is selected from HEK cells, CHO cells, COS cells and yeast cells. The yeast cell can be Saccharomyces cerevisiae and the insect cell can be an Sf9 cell.

  The invention also binds to RGM, comprising culturing host cells as defined herein in media under conditions sufficient to produce a binding protein capable of binding to RGM. Also provided is a method of producing a protein capable of

  The invention also relates to proteins produced according to this method.

The present invention also provides
(A) a formulation (this formulation comprises a crystallization product protein as defined herein) and ingredients;
(B) at least one polymeric carrier;
Also provided is a composition for the release of a binding protein.

  This polymeric carrier is poly (acrylic acid), poly (cyanoacrylate), poly (amino acid), poly (anhydride), poly (depsipeptide), poly (ester), poly (lactic acid), poly (lactic acid-glycol) Acid) copolymer or PLGA, poly (b-hydroxybutyrate), poly (caprolactone), poly (dioxanone); poly (ethylene glycol), poly ((hydroxypropyl) methacrylamide, poly [(organo) phosphazene], poly ( Ortho ester), poly (vinyl alcohol), poly (vinyl pyrrolidone), maleic anhydride-alkyl vinyl ether copolymer, pluronic polyol, albumin, alginate, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharide, glucose It may be selected from one or more of the group consisting of licaminoglycans, sulfated polysaccharides, mixtures thereof and copolymers.

  This component may be selected from the group consisting of albumin, sucrose, trehalose, lactitol, gelatin, hydroxypropyl-β-cyclodextrin, methoxypolyethylene glycol and polyethylene glycol.

  According to another aspect, the present invention provides a method for treating a mammal comprising administering to the mammal an effective amount of a composition as defined herein.

  According to another aspect, the present invention provides a pharmaceutical composition comprising a product (particularly a binding protein, construct or conjugate as described herein above) and a pharmaceutically acceptable carrier. provide.

  This pharmaceutically acceptable carrier can function as a useful adjuvant to improve absorption or dispersion of the binding protein.

  For example, the adjuvant is hyaluronidase.

  According to another embodiment, the formulation further comprises at least one additional therapeutic agent for treating a disease in which RGM activity is detrimental. For example, the agent is a therapeutic agent, contrast agent, cytotoxic agent, angiogenesis inhibitor; kinase inhibitor; costimulatory molecule blocker; adhesion molecule blocker; anti-cytokine antibody or functional fragment thereof; methotrexate; cyclosporine; rapamycin; Detectable label or reporter; TNF antagonist; anti-rheumatic agent; muscle relaxant, anesthetic, nonsteroidal anti-inflammatory drug (NSAID), analgesic, anesthetic, sedative, local anesthetic, neuromuscular blocker, antibacterial agent, Psoriasis drug, corticosteroid, anabolic steroid, erythropoietin, immunization, immunoglobulin, immunosuppressant, growth hormone, hormone supplement, radiopharmaceutical, antidepressant, antipsychotic, stimulant, asthma drug, beta agonist , Inhaled steroids, epinephrine or analogs, cytokines It is selected from the group consisting of fine cytokine antagonist.

  The present invention also provides that at least one product (particularly a binding protein, construct or conjugate, as described hereinabove) and human RGM A, such that at least one human RGM A activity is reduced. And a method for reducing human RGM A activity comprising contacting.

  The present invention also provides products of the invention (especially binding proteins, constructs or conjugates, as described herein above) to subjects in need of reducing hRGM A binding to the neogenin receptor. It also relates to a method for reducing hRGM A binding to a neogenin receptor in said subject comprising the step of administering.

  The present invention also provides a subject of the present invention (especially a product of the present invention (especially for hRGMA A binding to bone morphogenetic protein-2 and / or bone morphogenetic protein-4) Bone morphogenetic protein-2 and / or bone morphogenetic protein-4 (BMP-2 and BMP-4) in the subject comprising administering a binding protein, construct or conjugate as described hereinabove. Also relates to a method for reducing hRGM A binding to.

  The invention also includes administering a product of the invention (especially a binding protein, construct or conjugate, as described herein above) alone or in combination with other therapeutic agents, It also relates to a method for treating a subject for a disease associated with RGM A activity.

  The present invention also provides for the detrimental RGM A activity of the products of the present invention (particularly binding proteins, constructs or conjugates as described herein above) alone or in combination with other therapeutic agents. And a method for reducing RGMA activity in a subject suffering from a disease in which RGMA activity is detrimental, comprising administering to a subject suffering from a disease that is.

  This disease is preferably amyotrophic lateral sclerosis, brachial plexus injury, brain injury (including traumatic brain injury), cerebral palsy, Guillain Valley, cerebral white matter atrophy, multiple sclerosis, post-polio Syndrome, spina bifida, spinal cord injury, spinal muscular atrophy, spinal tumor, stroke, transverse myelitis; dementia, senile dementia, mild cognitive impairment, Alzheimer-related dementia, Huntington's chorea, late-onset dyskinesia , Hyperactivity, common disease, Parkinson's disease, Steel Richard syndrome, Down syndrome, myasthenia gravis, neurotrauma, vascular amyloidosis, cerebral hemorrhage with amyloidosis I, encephalitis, acute confusion disorder, amyotrophic lateral sclerosis A neurological disease selected from the group consisting of glaucoma and Alzheimer's disease.

  Further specific aspects of the invention are described below.

an isolated binding protein that specifically interacts with at least one epitope of the hRGM A protein;
The isolated protein which is a monoclonal neutralizing antibody or an antigen-binding fragment thereof;
Said antigen-binding fragment comprising VH and VL domains;
the neutralizing antibody, which reduces the ability of hRGM A to bind to its receptor;
said neutralizing antibody capable of inhibiting hRGM A biological activity;
Said antibody recognizing an RGM A receptor selected from human, cynomolgus monkey, rat, chick, frog and fish;
The antibody recognizing an RGM A protein having 90% homology with amino acid sequence SEQ ID NO: 2;
The antibody, wherein the RGM A protein is encoded by a nucleic acid having 90% homology with the nucleic acid sequence SEQ ID NO: 1;
Having at least 90% amino acid sequence identity with a heavy chain variable region (VH region) comprising the sequence of SEQ ID NO: 9 or 34, or a sequence comprising a humanized, optionally further mutated form of this VH region. Said antibody;
The antibody having at least 90% amino acid sequence identity with a light chain variable region (VL region) comprising the sequence of SEQ ID NO: 10 or a sequence comprising a humanized, optionally further mutated form of this VL region ;
said antibody that binds to hRGM A (this antibody is glycosylated);
The antibody or antigen-binding fragment which is a mouse antibody, humanized antibody, fully human, chimeric antibody, antigen-binding fragment of humanized antibody or antigen-binding fragment of chimeric antibody;
The antibody or antigen-binding fragment, which is an antigen-binding fragment selected from the group consisting of a Fab fragment, Fab ′ fragment, F (ab ′) ₂ fragment and Fv fragment;
said monoclonal antibody that specifically binds to at least one epitope of hRGM A (the monoclonal antibody is a monoclonal antibody secreted by a hybridoma cell line as described herein);
The monoclonal antibody, wherein binding results in inactivation of the interaction of hRGM A with its receptor;
said hybridoma cell line producing a monoclonal antibody that specifically binds to at least one epitope of hRGM A;
The hybridoma cell line wherein the hybridoma is selected from the group consisting of human, mouse, rat, sheep, pig, cow, goat and horse hybridoma;
Said monoclonal antibody wherein hRGM A is inactivated as a result of binding;
said hybridoma cell line producing a monoclonal antibody that specifically binds to at least one epitope of hRGM A;
The hybridoma cell line wherein the hybridoma is selected from the group consisting of human, mouse, rat, sheep, pig, cow, goat and horse hybridoma;
a) binds to mammalian RGM A with an affinity below the nM range;
b) functionally antagonize RGM A activity in vitro in a neurite outgrowth assay with an efficiency of μm, nM or less;
c) inducing germination in vivo in an optic nerve destruction model;
d) inducing germination in vivo in a spinal cord injury model;
e) reducing in vivo experimental spinal cord injury by promoting regenerative growth of damaged nerve fibers; or f) reducing in vivo experimental spinal cord injury by promoting synaptogenesis. Said monoclonal neutralizing antibody or antigen-binding fragment thereof having at least one property selected from the group;
An isolated nucleic acid encoding the monoclonal neutralizing antibody or antigen-binding fragment;
A vector containing the isolated nucleic acid;
pct; pTT; pTT3; pEFBOS; pBV; pJV; the vector selected from the group consisting of pHybE and pBJ;
A host cell transformed with the vector, in a form selected from the group consisting of protist cells, animal cells, plant cells and fungal cells;
A host cell, wherein the animal cell is a mammalian cell selected from the group consisting of HEK293, CHO and COS;
25. A host cell transformed with the vector of claim 24 which is a eukaryotic cell;
a method of producing a binding protein that binds to hRGM A, comprising culturing host cells in a medium under conditions sufficient to produce a binding protein that binds to hRGM A;
A pharmaceutical composition comprising the monoclonal antibody or antigen-binding portion and a pharmaceutically acceptable carrier;
A method for reducing hRGM A binding to a neogenin receptor in a subject comprising administering said antibody to a subject in need of decreasing hRGM A binding to a neogenin receptor;
Bone morphogenetic protein in a subject comprising administering said antibody to a subject in need of reducing hRGM A binding to bone morphogenetic protein-2 and bone morphogenetic protein-4 (BMP-2 and BMP-4) -2 and methods for reducing hRGM A binding to bone morphogenetic protein-4 (BMP-2 and BMP-4);
A method of treating a subject against a disease associated with RGM A activity comprising administering the antibody described above alone or in combination with other therapeutic agents;
A method for reducing RGM A activity in a subject comprising administering said antibody, alone or in combination with other therapeutic agents, to a subject suffering from a disease in which RGM A activity is detrimental;
Said antibody comprising at least one VH region comprising an amino acid sequence selected from SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, 42 and 43;
The antibody comprising at least one VL region comprising an amino acid sequence selected from SEQ ID NOs: 44, 45 and 46;
The antibody further modified by 1 to 5 mutations in the VH or VL sequence;
The antibody wherein the mutation is selected from a framework back mutation and a mutation of a vernier and VH / VL contact residue;
Similarly, any teaching or reference to SEQ ID NO: 34 as disclosed herein applies to SEQ ID NO: 9.

FIG. 1A shows that monoclonal antibodies bind to hRGM A in an ELISA assay. FIG. 1B shows that the monoclonal antibody binds to hRGM A expressed in HEK293 cells. FIG. 1C shows that the monoclonal antibody binds to rat RGM A expressed in HEK293 cells. FIG. 2 shows that full-length RGM A binds to neogenin. MAB 5F9 inhibits binding of full-length, fc-linked hRGM A to neogenin. FIG. 3 shows that full-length RGM A binds to BMP-4. MAB 5F9 inhibits the binding of fc-linked full length hRGM A fragment (47-422) to BMP-4. FIG. 4 shows that RGM A fragment 0 binds to BMP-4. MAB 5F9 inhibits the binding of fc-linked hRGM A fragment 0 (47-168) to BMP-4. FIG. 5 shows that full-length RGM A binds to BMP-2. MAB 5F9 inhibits the binding of fc-linked full-length hRGM A fragment (47-422) to BMP-2. FIG. 6 is a combination of photomicrographs showing neutralization of RGM A fragment by mAb 5F9 in Ntera cell neurite outgrowth assay. MAB 5F9 neutralizes the elongation inhibitory activity of the fc-bound potent hRGM A inhibitor fragment in the neurite outgrowth assay using human Ntera aggregates. A. B. in a control culture, laminin. C. in laminin-hRGM A fragment (47-168) substrate. -E. Ntera neurons in laminin-hRGM A fragment (47-168) substrate in the presence of 0.1 μg / mL MAB 5F9 (C), 1 μg / mL MAB 5F9 (D.), 10 μg / mL MAB 5F9 (E.) Growth. FIG. 7 shows quantitative analysis of NTera2 assay results. MAB 5F9 dose-dependently neutralizes the elongation inhibitory activity of fc-bound potent hRGM A inhibitor fragments (fragments 0, 47-168) in a neurite outgrowth assay using human Ntera aggregates. FIG. 8 shows the quantitative analysis of the SH-SY5Y stripe assay. MAB 5F9 neutralizes the repulsion induced by stripes consisting of full-length human RGM A in human SH-SY5Y neuronal cells in a striped membrane carpet. Neurons tend to avoid RGM A stripes in the absence of MAB 5F9 (A) or in the presence of low MAB concentrations SH-SY5Y. This behavior is reversed by increasing the concentration of MAB 5F9. (B to D) At the maximum MAB concentration (10 μg / mL) (E), SH-SY5Y neurons show strong selectivity for the RGM A stripe compared to the collagen I stripe. FIG. 8 shows the quantitative analysis of the SH-SY5Y stripe assay. MAB 5F9 neutralizes the repulsion induced by stripes consisting of full-length human RGM A in human SH-SY5Y neuronal cells in a striped membrane carpet. Neurons tend to avoid RGM A stripes in the absence of MAB 5F9 (A) or in the presence of low MAB concentrations SH-SY5Y. This behavior is reversed by increasing the concentration of MAB 5F9. (B to D) At the maximum MAB concentration (10 μg / mL) (E), SH-SY5Y neurons show strong selectivity for the RGM A stripe compared to the collagen I stripe. FIG. 8 shows the quantitative analysis of the SH-SY5Y stripe assay. MAB 5F9 neutralizes the repulsion induced by stripes consisting of full-length human RGM A in human SH-SY5Y neuronal cells in a striped membrane carpet. Neurons tend to avoid RGM A stripes in the absence of MAB 5F9 (A) or in the presence of low MAB concentrations SH-SY5Y. This behavior is reversed by increasing the concentration of MAB 5F9. (B to D) At the maximum MAB concentration (10 μg / mL) (E), SH-SY5Y neurons show strong selectivity for the RGM A stripe compared to the collagen I stripe. FIG. 8 shows the quantitative analysis of the SH-SY5Y stripe assay. MAB 5F9 neutralizes the repulsion induced by stripes consisting of full-length human RGM A in human SH-SY5Y neuronal cells in a striped membrane carpet. Neurons tend to avoid RGM A stripes in the absence of MAB 5F9 (A) or in the presence of low MAB concentrations SH-SY5Y. This behavior is reversed by increasing the concentration of MAB 5F9. (B to D) At the maximum MAB concentration (10 μg / mL) (E), SH-SY5Y neurons show strong selectivity for the RGM A stripe compared to the collagen I stripe. FIG. 8 shows the quantitative analysis of the SH-SY5Y stripe assay. MAB 5F9 neutralizes the repulsion induced by stripes consisting of full-length human RGM A in human SH-SY5Y neuronal cells in a striped membrane carpet. Neurons tend to avoid RGM A stripes in the absence of MAB 5F9 (A) or in the presence of low MAB concentrations SH-SY5Y. This behavior is reversed by increasing the concentration of MAB 5F9. (B to D) At the maximum MAB concentration (10 μg / mL) (E), SH-SY5Y neurons show strong selectivity for the RGM A stripe compared to the collagen I stripe. FIG. 9 summarizes the quantitative analysis of mAB 5F9 and 8D1 binding properties. 9 and 8D1 are evaluated at various concentrations in MAB5FhRGM A-neogenin, hRGM A-BMP-2 and hRGM A-BMP-4 binding assays. FIG. 10 shows the neutralizing activity of the humanized 5F9 antibodies (h5F9.21, h5F9.23, h5F9.25) against the chemical repulsion activity of hRGM A in the SH-SY5Y chemotaxis assay. FIG. 11 shows the in vivo nerve regeneration activity of 5F9 topical application in an animal model of optic nerve injury. Topical application of MAB 5F9 neutralizes RGM A and stimulates regenerative elongation of impaired optic nerve axons in an optic nerve destruction rat animal model. In 5F9 treated animals (A), in contrast to the control MAB8D1 (B), which does not bind to rat RGM A, many GAP-43 positive fibers extend beyond the site of destruction. 12A and 12B show a quantitative analysis of 5F9 topical application in an animal model of optic nerve injury. (A) 5F9 significantly increased the number of regenerated GAP-43 positive fibers, but not the control MAB8D1. In 5F9 treated animals, significantly more fibers (p <0.05) were observed at distances of 200 μm, 400 μm and 600 μm, and at 1200 μm fibers were only seen in 5F9 treated animals and not in the subject animals, (B) 5F9 significantly increased the GAP-43 positive area at the optic nerve injury site compared to control antibody 8D1 and vehicle control PBS. Regeneration extension area (GAP-43 positive area) was measured using Axiovision software (Zeiss). FIG. 13 shows the in vivo nerve regeneration activity of 5F9 systemic application in an animal model of optic nerve injury. Animals were treated with 5F9 on days 0 and 21 at 2 mg / kg and 10 mg / kg, respectively. Antibody or vehicle was administered intraperitoneally or intravenously. Composite image of rat optic nerve. In 5F9 treated animals (A), many GAP-43 positive fibers extend beyond the site of destruction, in contrast to PBS treated control animals (B). The destruction site is at the left end, and the regenerated fibers are stained with an antibody against GAP-43. Many fibers are observed at the upper and lower edges of the optic nerve of 5F9-treated animals, but not in PBS animals. FIG. 14A shows a quantitative analysis of 5F9 whole body application in an animal model of optic nerve injury. FIG. 14B shows a quantitative analysis of 5F9 whole body application in an animal model of optic nerve injury. FIG. 15 shows the in vivo remyelination activity of 5F9 systemic application in an animal model of optic nerve injury. Animals were treated with 5F9 on days 0 and 21 at 2 mg / kg and 10 mg / kg, respectively. Antibody or vehicle was administered intraperitoneally or intravenously. Composite image of rat optic nerve. Myelin formation is visualized using an antibody against the myelin marker myelin basic protein MBP. The site of destruction is in the middle of the complex nerve and this area is not present in vehicle-treated control animals (A and B). In 5F9 treated animals (C and D), many MBP positive structures are observed in the central region of the optic nerve (destructive center). FIG. 16 shows the quantitative effect on remyelination of systemic application of 5F9 in an animal model of optic nerve injury.

(Detailed description)
The present invention describes RGM A binding proteins, particularly monoclonal RGM A antibodies, particularly humanized monoclonal RGM A antibodies, or antigen-binding portions thereof, that bind to RGM A. Various aspects of the present application relate to antibodies and antibody fragments and pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such antibodies and fragments. Also included are methods of using the antibodies of the present application to detect human RGM A, to neutralize human RGM and / or human RGM A activity, either in vivo or in vitro, and to control gene expression. Included in the invention.

1. General Definitions Unless defined otherwise herein, scientific and technical terms used in connection with the present invention have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of these terms are clear, but in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionaries or ancillary definitions. . Further, unless otherwise required by content, singular terms include pluralities and plural terms include singularities. In this application, the use of “or” means “and / or” unless stated otherwise. Furthermore, the use of the term “including” and other forms such as “includes” and “included” is not limiting. Also, unless stated otherwise, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise a plurality of subunits.

  In general, the nomenclature and techniques used herein in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization are described in this document. It is well known in the technical field and commonly used. The methods and techniques of the present invention are generally described in various general and more specific references cited and discussed throughout this application, according to conventional methods well known in the art, unless otherwise indicated. Done. Enzymatic reactions and purification techniques are performed according to the manufacturer's specifications, as commonly performed in the art, or as described herein. The nomenclature and their testing methods and techniques used herein in connection with analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry are well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, drug formulation, formulation and delivery and patient treatment.

  In order that the present invention may be more readily understood, selected terms are defined below.

  The term “polypeptide” as used herein refers to any polymer chain of amino acids. The terms “peptide” and “protein” are used interchangeably with the term polypeptide and also refer to a polymer chain of amino acids. The term “polypeptide” encompasses native or artificial proteins, protein fragments and polypeptide analogs of a protein sequence. Polypeptides can be monomeric or polymeric.

  The term "isolated protein" or "isolated polypeptide" refers to a protein or polypeptide that is not associated with its naturally associated components in its native state, based on its origin or source of derivative. Is substantially free of other proteins from the same species; is expressed by cells from different species; or is not naturally occurring. In this way, a polypeptide that is chemically synthesized or synthesized in a cell line different from the cell from which it is naturally derived is “isolated” from its naturally associated components. The protein can also be made substantially free of naturally associated components by isolation using protein purification techniques well known in the art.

  The term “recover” as used herein refers to a chemical species, such as a polypeptide, that substantially eliminates naturally associated components by isolation, eg, using protein purification techniques well known in the art. Refers to processes that are not included in.

  “Human RGM A” (abbreviated herein as hRGM A), as used herein, refers to a 450 amino acid glycosylphosphatidyl-inositol (gpi) -anchored glycoprotein, It has been described as a neurite outgrowth depletion or neurite outgrowth inhibitor during the topographic projection (Stahl et al., Neuron 5: 735-43, 1990; Mueller, Molecular Basis of Axon Growth Pattern). H. Hujisawa, Japan Scientific Society Press, 215-229, 1997). The rgm gene family includes three different genes, two of which, rgma a and b, are expressed in the mammalian CNS, while the third member, rgmc, is expressed peripherally (Müller et al., Philos.Trans.R.Soc.Lon.Biol.Sci.361: 1513-29, 2006), where rgmc plays an important role in iron metabolism. The human RGM protein has 43% -50% sequence identity and the amino acid homology of human and rat RGM A is 89%. Human RGM protein does not have significant sequence homology with any other known protein. These are proline-rich proteins that contain an RGD region, have structural homology to the von Willebrand factor domain, and are cleaved at the N-terminal amino acid 168 by an unknown protease, resulting in a functionally active protein (Müller et al., Philos. Trans. R. Soc. London. Biol. Sci. 361: 1513-29, 2006).

In vitro, RGM A inhibits neurite outgrowth at picomolar concentrations by binding to neogenin, which has been identified as an RGM receptor (Rajagopalan et al., Nat Cell Biol .: 6 (8), 756-62, 2004). . Although neogenin was first described as a netrin-binding protein (Keino-Masu et al., Cell, 87 (2): 175-85, 1996), its affinity for netrin (K _d 2nM) was determined by RGM (K _d 0.2 nM) (Rajagopalan et al., Nat Cell Biol .: 6 (8), 756-62, 2004). This is an important finding because it has been reported that binding of netrin-1 to neogenin or to its related receptor DCC (deleted in colorectal cancer) stimulates neurite outgrowth rather than inhibiting it. (Braisted et al., J. Neurosci. 20: 5792-801, 2000).

  In addition to binding RGM A to neogenin and inducing inhibition of neurite outgrowth, binding of RGM A or B to bone morphogenetic proteins BMP-2 and BMP-4 can be a similar obstacle to successful nerve regeneration and functional recovery. (Müller et al., Philos. Trans. R. Soc. London. Biol. Sci. 361: 1513-29, 2006). Both classes of proteins (neogenin and BMP) have been reported to transduce RGM A neurite outgrowth inhibitory signals through two completely different and independent signaling circuits. Usually, expression of these BMP proteins is relatively low in most regions of the adult CNS, but in response to injury and injury, expression of some BMPs (eg, BMP-2, BMP-6, BMP-7) And accumulation is reported to rise rapidly (Lai et al., Neuroport 8: 2691-94, 1997; Martinez et al., Brain Res. 894: 1-11, 2001; Hall and Miller, J. Neurosci. Res. 76: 1-8, 2004; Setoguchi et al., Exp. Neurol. 189: 33-44, 2004). Furthermore, in a multiple sclerosis model, an experimental autoimmune encephalomyelitis (EAE) model, BMP-4, BMP-6 and BMP-7 were up-regulated in the mouse spinal cord (Ara et al., J. Neurosci. Res. 86: 125-35, 2008). BMP-2 has been reported to inhibit neurite outgrowth by binding to cell surface RGM A, BMP-receptors I and II and by directly activating LIM-kinase (Matsuura et al., Biochem Biophys Res Commun., 360: 868-73, 2007), and therefore, it is expected that functional recovery after CNS injury will be further facilitated by blocking RGM A-BMP-2 interaction.

  As mentioned above, spinal cord injured rats and brain-injured humans show extensive accumulation of cellular RGM at the site of injury, and the staining pattern of RGM A in rats at the site of spinal cord lesions is determined by total RGM antibodies in humans. This is very similar to the staining, suggesting that most of the total RGM staining in humans is related to RGM A localization rather than RGM B localization (Schwab et al., Arch. Neurol). 62: 1561-8, 2005a; Schwab et al., Eur. J. Neurosci. 21: 1569-76, 2005b; Hata et al., J. Cell. Biol. 173: 47-58, 2006). Total RGM staining (RGM A and B immunoreactivity) was detected in white matter fibers, oligodendrocytes, few neuronal perinuclear bodies, some vascular smooth muscles and few endothelial cells in healthy human brain . Astrocyte staining was not observed. The RGM staining pattern of adult healthy human brain is very similar to that observed in the adult rat spinal cord (Schwab et al., Eur. J. Neurosci. 21: 1569-76, 2005b; Hata et al., J. Cell. Biol. 173: 47-58, 2006).

  Based on the accumulation of RGM A at the lesion in brain and spinal cord injuries, and because of its cellular neurite outgrowth inhibitory activity, this protein exerts neurite outgrowth inhibitory activity and is associated with at least one epitope of human RGM A. It is expected that neutralization by the binding antibody or antigen-binding fragment thereof will result in improved regrowth of damaged nerve fibers and promote functional recovery in indicators characterized by nerve fiber damage and RGM accumulation Is done.

  Unless otherwise noted, the term “RGM A” also refers to RGM A molecules isolated or obtained from other species, eg, rodents such as mice or rats, specifically “ Also included is a rat-derived molecule called "Rat RGM A".

  “Biological activity” as used herein refers to all native biological properties of RGM A as defined herein.

  The term “specific binding” or “specifically bind” as used herein refers to the interaction of an antibody, protein or peptide with a second chemical species, where the interaction is Means dependent on the presence of a particular structure in a species (eg, an “antigenic determinant” or “epitope” as defined below); for example, an antibody is generally a protein rather than a protein Recognize and bind to structures. When an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A) binds to the antibody in a reaction containing labeled “A” and the antibody. The amount of labeled A is reduced.

  The term “antibody” as used herein broadly refers to any immunoglobulin composed of four polypeptide chains, two heavy (H) chains and two light (L) chains. (Ig) molecule or any functional fragment, mutant, variant or derivative thereof, which retains the essential epitope binding properties of the Ig molecule. Such mutant, variant or derived antibody forms are known in the art. This non-limiting embodiment is discussed below. An antibody is said to be “bindable” to a molecule when it can specifically react with the molecule, thereby binding the molecule to the antibody.

  "Monoclonal antibody" as used herein refers to an antibody molecule having a common heavy and light chain amino acid sequence relative to a "polyclonal" antibody preparation containing a mixture of various antibodies. The preparation of Monoclonal antibodies may be produced by hybridoma technology, such as phage, bacteria, yeast or ribosome display, as well as hybridoma-derived antibodies (eg, standard Kohler and Milstein hybridoma methods ((1975) Nature 256: 495-497)). Antibodies secreted by the prepared hybridomas).

  In full-length antibodies, each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. VH and VL regions can be further classified into hypervariable regions called complementarity determining regions (CDRs) interspersed with regions called framework regions (FR) that are more conserved. Each VH and VL is composed of three CDRs and four FRs arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus. The immunoglobulin molecules can be of any type (eg, IgG, IgE, IgM, IgD, IgA and IgY), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

The term “antigen-binding portion” or “antigen-binding fragment” of an antibody (or simply “antibody portion” or “antibody fragment”), as used herein, is specific for an antigen (eg, hRGM A). Refers to one or more fragments of an antibody that retain the ability to bind to. It has been shown that the antigen-binding function of an antibody can be exerted by fragments of a full-length antibody. Such antibody embodiments can also be bispecific, bispecific or multispecific forms that specifically bind to two or more different antigens. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) Fab fragments (monovalent fragments consisting of VL, VH, CL and CH1 domains), (ii) F (ab ′) ₂ fragments (a bivalent fragment containing 2 Fab fragments linked by a disulfide bridge at the hinge region), (iii) an Fd fragment consisting of VH and CH1 domains, (iv) consisting of the VL and VH domains of a single arm of the antibody Fv fragment, (v) a dAb fragment containing one variable domain (Ward et al. (1989) Nature 341: 544-546, Winter et al., PCT Publication WO 90 / 05144A1 (incorporated herein by reference)) and ( vi) An isolated complementarity determining region (CDR) is included. In addition, the two domains of the Fv fragment, VL and VH, are encoded by separate genes, but these are as a single protein chain in which the VL and VH regions pair to form a monovalent molecule. Can be linked using recombinant methods (known as single chain Fv (scFv). For example, Bird et al. (1988) Science 242: 423-426; and Huston et al. ( 1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883. Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody, such as diabodies. Other forms of single chain antibodies are also included: diabodies are on polypeptide chains with a single VH and VL domain. Use linkers that are expressed but are too short to allow pairing between the two domains on the same strand, thereby forcing these domains to be complementary to the complementary domains of another strand Bivalent bispecific antibodies that pair and create two antigen binding sites (eg, Holliger, P. et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak R J. et al. (1994) Structure 2: 1121-1123) Such antibody binding moieties are known in the art (Edited by Kontermann and Dubel, Antibody Engineering (2001) Springer-Verlag. New York.). 790 pp. (ISBN 3-540-41354-5).

  The term “antibody construct” as used herein refers to a polypeptide comprising one or more antigen-binding portions of the invention linked to a linker polypeptide or an immunoglobulin constant domain. A linker polypeptide comprises two or more amino acid residues linked by peptide bonds and is used to link one or more antigen binding moieties. Such linker polypeptides are well known in the art (see, eg, Holliger, P. et al. (1993) Proc. Natl. Acad. Sci USA 90: 6444-6448; Poljak, RJ et al. (1994) Structure). 2: 1121-1123). An immunoglobulin constant domain refers to a heavy or light chain constant domain. Human IgG heavy and light chain constant domain amino acid sequences are known in the art and are shown in Table 2.

Still further, the antibody or antigen-binding portion thereof can be part of a larger immunoadhesion molecule formed by covalent or non-covalent attachment of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of the streptavidin core region to generate tetrameric scFv molecules (Kipriyanov, SM et al. (1995) Human Antibodies and Hybridomas 6: 93-101) and two. Includes the use of cysteine residues, marker peptides and C-terminal polyhistidine tags (Kipriyanov, SM et al. (1994) Mol. Immunol. 31: 1047-1058) to generate valent and biotinylated scFv molecules . Antibody portions, such as Fab and F (ab ′) ₂ fragments, can be prepared from the whole antibody using conventional techniques such as papain or pepsin digestion of the whole antibody, respectively. In addition, antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.

  An “isolated antibody” as used herein is intended to refer to an antibody that is substantially free of other antibodies with different antigen specificities (eg, specifically binds to hRGM A). Isolated antibodies are substantially free of antibodies that specifically bind antigens other than hRGM A). However, an isolated antibody that specifically binds hRGM A may have cross-reactivity with other antigens such as RGM A molecules from other species. Furthermore, the isolated antibody may be substantially free from other cellular substances and / or chemical substances.

  As used herein, the term “human antibody” is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present invention may contain amino acid residues that are not encoded by human germline immunoglobulin sequences in, eg, CDRs, particularly CDR3 (eg, by in vitro random or site-directed mutagenesis or in vivo somatic cells. Mutations introduced by mutations). However, the term “human antibody” as used herein includes an antibody in which a CDR sequence from the germline of another mammalian species such as a mouse is grafted onto a human framework sequence. Make it not exist.

  The term “recombinant human antibody” as used herein refers to an antibody (detailed below), recombinant, combinatorial human antibody library expressed using a recombinant expression vector transfected into a host cell. (Hoogenboom HR (1997) TIB Tech. 15: 62-70; Azzay H. and Highsmith WE (2002) Clin. Biochem. 35: 425-445; Gavilondo J.V. And Larrick JW (2002) BioTechniques 29: 128-145; Hoogenboom H. and Chames P. (2000) Immunology Today 21: 371-378), against human immunoglobulin genes. Antibodies isolated from animals that are transgenic (eg, mice) (eg, Taylor, LD et al. (1992) Nucl. Acids Res. 20: 6287-6295; Kellermann SA and Green LL (2002). ) Current Opinion in Biotechnology 13: 593-597; Little M. et al. (2000) Immunology Today 21: 364-370) or any other means including splicing of human immunoglobulin gene sequences to other DNA sequences It includes all human antibodies prepared, expressed, produced or isolated by recombinant means, such as antibodies that are expressed, expressed, produced or isolated. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subject to in vitro mutagenesis (or in vivo somatic mutagenesis if animals that are transgenic for human Ig sequences are used), Thus, the amino acid sequences of the VH and VL regions of the recombinant antibody are derived from and related to human germline VH and VL sequences, sequences that cannot occur naturally within the human antibody germline repertoire in vivo. It is.

  The term “chimeric antibody” refers to a heavy and light chain variable region sequence derived from one species and a constant region derived from another species, such as an antibody having a mouse heavy and light chain variable region linked to a human constant region. Refers to an antibody comprising a sequence. Chimeric antibodies can be made through recombinant molecular biology techniques or can be physically linked together.

  The term “CDR grafted antibody” refers to heavy and light chains derived from certain species, such as antibodies having murine heavy and light chain variable region sequences in which one or more of the murine CDRs (eg, CDR3) are replaced with human CDR sequences. Refers to an antibody comprising a chain variable region sequence, wherein one or more sequences of the CDR regions of VH and / or VL are replaced with another species CDR sequence.

  The terms “Kabat numbering”, “Kabat definition” and “Kabat labeling” are used interchangeably herein. These terms are recognized in the art and are more variable (ie, hypervariable) amino acid residues than other amino acid residues in the heavy and light chain variable regions of an antibody or antigen binding portion thereof. (Kabat et al. (1971) Ann. NY Acad, Sci. 190: 382-391 and Kabat, EA et al. (1991) Sequences of Immunological Interest, 5th edition, US. Department of Health and Human Services, NIH Publication No. 91-3242). For the heavy chain variable region, the hypervariable region ranges from amino acid positions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, and amino acid positions 95 to 102 for CDR3. For the light chain variable region, the hypervariable region ranges from amino acid positions 24 to 34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acid positions 89 to 97 for CDR3.

  As used herein, the terms “acceptor” and “acceptor antibody” refer to at least 80%, at least 85%, at least 90%, at least 95%, at least 98 of one or more amino acid sequences of a framework region. Refers to an antibody or nucleic acid sequence that gives or encodes% or 100%. In certain embodiments, the term “acceptor” refers to an antibody amino acid or nucleic acid sequence that provides or encodes a constant region. In yet another embodiment, the term “acceptor” refers to an antibody amino acid or nucleic acid sequence that provides or encodes one or more of a framework region and a constant region. In a specific embodiment, the term “acceptor” provides at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98% or 100% of one or more amino acid sequences of the framework region. Refers to or encodes a human antibody amino acid or nucleic acid sequence. According to this embodiment, the acceptor may contain at least 1, at least 2, at least 3, at least 4, at least 5, or at least 10 amino acid residues that do not occur at one or more specific positions of the human antibody. Acceptor framework regions and / or acceptor constant regions are derived from, for example, germline antibody genes, mature antibody genes, functional antibodies (eg, antibodies well known in the art, antibodies under development or commercially available antibodies). Can be or can be derived from them.

  As used herein, the term “CDR” refers to the complementarity determining region within antibody variable sequences. There are three CDRs in each of the heavy and light chain variable regions, referred to as CDR1, CDR2 and CDR3 for each of the variable regions. The term “CDR set” as used herein refers to a group of three CDRs that occur in one variable region that can bind an antigen. The correct boundaries for these CDRs are defined separately by the system. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987) and (1991)) is applicable to all variable regions of antibodies. In addition to providing a numbering system, it also provides clear residue boundaries that define three CDRs, which can be referred to as Kabat CDRs, Chothia and co-workers (Chothia and Lesk, J. Mol. Biol 196: 901-917 (1987) and Chothia et al., Nature 342: 877-883 (1989)) are also highly diverse at the amino acid sequence level. Nevertheless, it has been found that certain portions within the Kabat CDR have approximately the same peptide backbone structure, these portions being named L1, L2 and L3 or H1, H2 and H3. (Where "L" and "H" represent the light and heavy chain regions, respectively.) These regions can be referred to as Chothia CDRs, which have boundaries that overlap with the Kabat CDRs. Other boundaries defining CDRs that overlap with Kabat CDRs are described by Padlan (FASEB J. 9: 133-139 (1995)) and MacCallum (J Mol. Biol. 262 (5): 732-45 (1996)). Yet another CDR boundary definition is exactly one of the above systems. Considering the expected or experimental findings that specific residues or groups of residues or even the entire CDR may not significantly affect antigen binding, although they may not be followed, they can be shortened or lengthened Regardless of whether or not, it still overlaps with the Kabat CDR, although the methods used herein may use CDRs defined according to any of these systems, preferred embodiments include Kabat or Uses CDRs defined in Chothia.

  As used herein, the term “canonical” residue refers to Chothia et al. (J. Mol. Biol. 196: 901-907 (1987); Chothia et al., J. Mol. Biol. 227. : 799 (1992), both of which are incorporated herein by reference) to refer to residues in the CDRs or frameworks that define a particular canonical CDR structure. According to Chothia et al., An important part of the CDRs of many antibodies have nearly identical peptide backbone structures, albeit very diverse at the amino acid sequence level. Each canonical structure primarily refers to a set of peptide backbone torsion angles relative to adjacent segments of amino acid residues that form a loop.

  As used herein, the terms “donor” and “donor antibody” refer to an antibody that provides one or more CDRs. In preferred embodiments, the donor antibody is an antibody from a different species than the antibody from which the framework region is obtained or derived. With respect to humanized antibodies, the term “donor antibody” refers to a non-human antibody that provides one or more CDRs.

  As used herein, the term “framework” or “framework sequence” refers to the remaining sequence of the variable region minus the CDRs. Since the exact definition of CDR sequences can be determined by different systems, the meaning of framework sequences follows correspondingly different interpretations. The six CDRs (light chain CDR-L1, -L2 and -L3 and heavy chain CDR-H1, -H2 and -H3) also contain light chain and heavy chain framework regions in four sub-chains in each chain. Divided into regions (FR1, FR2, FR3 and FR4), where CDR1 is located between FR1 and FR2, CDR2 is located between FR2 and FR3, and CDR3 is between FR3 and FR4. Located in. When specified by others without specifying a particular subregion as FR1, FR2, FR3 or FR4, the framework region represents the combined FR ′ within the variable region of one natural immunoglobulin chain. . As used herein, FR (singular) represents one of the four subregions, and FR (plural) represents two or more of the four subregions that make up the framework region.

  Human heavy and light chain acceptor sequences are known in the art. In certain embodiments of the invention, the human heavy and light chain acceptor sequences are selected from the sequences set forth in Table 3 and Table 4. Various combinations for human framework sequences FR1 to FR4 are described in these tables.

  As used herein, the term “germline antibody gene” or “gene fragment” is not subject to a maturation process that results in gene rearrangement and mutation to the expression of a particular immunoglobulin. Refers to immunoglobulin sequences encoded by non-lymphoid cells (eg, Shapiro et al., Crit. Rev. Immunol. 22 (3): 183-200 (2002); Marchalonis et al., Adv Exp Med Biol. 484: 13-30). (2001)). One of the advantages provided by the various embodiments of the present invention is that germline antibody genes are more likely to preserve the characteristics of an individual's essential amino acid sequence structure in a species than mature antibody genes, Therefore, it is due to the recognition that when used therapeutically in that species, it is unlikely to be recognized as originating from a foreign source.

  As used herein, the term “key” residue refers to a certain region within the variable region that has a greater impact on the binding specificity and / or affinity of an antibody, particularly a humanized antibody. Refers to the residue. Key residues include, but are not limited to: residues adjacent to CDRs, potential glycosylation sites (which can be either N- or O-glycosylation sites), rare residues Group, residue capable of interacting with antigen, residue capable of interacting with CDR, canonical residue, contact residue between heavy chain variable region and light chain variable region, residue in vernier zone and variable One or more of the residues in the region overlapping between the Chothia definition of heavy chain CDR1 and the Kabat definition of the first heavy chain framework is included.

  The term “humanized antibody” generally refers to an antibody comprising heavy and light chain variable region sequences from a non-human species (eg, mouse), but where at least a portion of the VH and / or VL sequences. Have been modified to be more “near human”, ie, more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody, in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding non-human CDR sequences.

  In particular, the term “humanized antibody” as used herein immunospecifically binds to an antigen of interest and includes a framework (FR) region having substantially the amino acid sequence of a human antibody and An antibody or variant, derivative, analog or fragment thereof, comprising a complementarity determining region (CDR) having the amino acid sequence of a substantially non-human antibody. As used herein, the term “substantially” with respect to a CDR is the amino acid sequence of the non-human antibody CDR and at least 50, 55, 60, 65, 70, 75 or 80%, preferably at least 85%. , CDRs having an amino acid sequence that is at least 90%, at least 95%, at least 98% or at least 99% identical. A humanized antibody is one in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (ie, donor antibody) and all or substantially all of the framework regions are of human immunoglobulin consensus sequence. , Including substantially all of the variable domains (Fab, Fab ′, F (ab ′) 2, FabC, Fv), at least one and usually two. Preferably, the humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), usually that of a human immunoglobulin. In certain embodiments, a humanized antibody contains both the light chain as well as at least the variable domain of a heavy chain. The antibody may also comprise the CH1, hinge, CH2, CH3 and CH4 regions of the heavy chain. In certain embodiments, a humanized antibody contains only a humanized light chain. In certain embodiments, the humanized antibody contains only humanized heavy chains. In certain embodiments, a humanized antibody contains only the humanized variable domain of the light chain and / or the humanized heavy chain.

  Humanized antibodies can be from any class of immunoglobulins, including IgY, IgM, IgG, IgD, IgA, and IgE, and any isotype (including but not limited to IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4). You can choose. Humanized antibodies can comprise sequences from multiple classes or isotypes, and techniques that are well known in the art can be used to select specific constant domains to optimize the desired effector function.

  The framework and CDR regions of a humanized antibody need not correspond exactly to the parent sequence, eg, so that no CDR or framework residues at that site correspond to either the donor antibody or the consensus framework, Mutagenesis can be performed on the donor antibody CDR or consensus framework by substitution, insertion and / or deletion of at least one amino acid residue. However, in preferred embodiments, such mutations are not extensive. Usually, at least 50, 55, 60, 65, 70, 75 or 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95% of the humanized antibody residues are parental FR and CDR sequences Corresponds to As used herein, the term “consensus framework” refers to a framework region in a consensus immunoglobulin sequence. As used herein, the term “consensus immunoglobulin sequence” refers to a sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related immunoglobulin sequences (eg, Winnaker, From Gene to Clones (see Verlagsgesellschaft, Weinheim, Germany 1987) In the immunoglobulin family, each position in the consensus sequence has the most frequently occurring amino acid at that position in the family. Can appear in the consensus sequence.

  As used herein, “vernier” zones are as described by Foote and Winter (1992, J. Mol. Biol. 224: 487-499, incorporated herein by reference). In addition, it refers to a subset of framework residues that can tailor the CDR structure and fine-tune the fit to the antigen. Vernier zone residues form the layer underlying the CDRs and can affect CDR structure and antibody affinity.

  The term “inhibition of binding” of RGM to one of its receptors, as used herein, is a partial (eg, about 20%, 40%, 60%) of this receptor binding activity. 80%, 85%, 90%, 95% or more) or complete reduction. This “inhibition of binding” can be determined by any suitable method available in the art, preferably by any method as exemplified herein, eg, an ELISA-based binding assay.

  As used herein, the term “neutralization” refers to neutralization of the biological activity of a target protein when the binding protein specifically binds to the target protein. Neutralization can be the result of various means of binding of the binding protein to the target. For example, neutralization can be caused by binding of the binding protein in a region of the target that does not affect receptor binding to the target molecule. Alternatively, binding of the binding protein can result in blocking receptor binding to the target, which ultimately neutralizes the target protein activity. Each of these various functions can occur in accordance with the present invention. Preferably, the neutralizing binding protein is a neutralizing antibody that, as a result of its binding to hRGM A, neutralizes the biological activity of hRGM A. Preferably, the neutralizing binding protein binds to hRGM A and biologically reduces the activity of hRGM A by at least about 20%, 40%, 60%, 80%, 85% or more. By measuring one or more indicators of hRGM A biological activity well known in the art, neutralization of hRGM A biological activity by neutralizing binding proteins can be assessed. For example, neutralization of hRGM A reverses inhibition in the Ntera nerve elongation assay (see Example 3 below). The Ntera neurite growth assay addresses the inhibition of neurite outgrowth. In the absence of inhibitory RGM A protein or fragment and in the presence of the elongation stimulating substrate laminin, neuronal NTera aggregation represents a large and dense network of elongating neurites. RGM A or RGM A fragments inhibit neurite outgrowth, resulting in shorter neurites and a reduced number of them. A function-blocking RGM A antagonist or MAB (such as MAB 5F9) is a potent fc-linked hRGM A light chain fragment (amino acids 47-168) of human RGM A protein in neurite outgrowth assays with aggregates of differentiated human NTera neurons. Neutralization inhibition activity was neutralized, resulting in longer neurites and a significant increase in the number.

  “Neutralizing monoclonal antibody” as used herein refers to a preparation of an antibody molecule whose binding to a specific antigen can compete with and inhibit the binding of a natural ligand to this antigen. Shall point to. In certain embodiments of the present application, the neutralizing antibodies of the present invention can compete with RGM A for binding to neogenin and / or to BMP-2 and / or BMP-4 and interfere with RGM A biological activity or function. Can do. In particular, the neutralizing antibodies of the present invention may bind to RGM A, interfere with binding to neogenin and / or to BMP-2 and / or BMP-4, and may interfere with RGM A biological activity or function. The term “activity” includes the binding specificity / affinity of an antibody to an antigen (eg, an anti-hRGM A antibody that binds to an RGM A antigen) and / or the neutralizing ability of an antibody (eg, its binding to RGM A is represented by hRGM An anti-hRGM A antibody that inhibits the biological activity of A (e.g., as measured in an hRGM A-neogenin binding assay, hRGM A-BMP-2 binding assay or hRGM A-BMP-4 binding assay as described in the experimental section below) )) And other activities are included.

  The biological activity of RGM A can also be said to control cell migration. A specific example of cell migration is neurite outgrowth, which is interrupted or inhibited by RGM A protein. Furthermore, RGM protein has been shown to modulate the activity of BMP-protein. The published examples herein are unilaterally the synergistic enhancing activity of the RGM protein in the BMP-pathway and the inhibitory activity of the RGM protein in the BMP-pathway (which controls iron metabolism, bone and cartilage regeneration) And is important in the CNS for remyelination and regeneration).

  The term “epitope” or “antigenic determinant” includes any polypeptide determinant capable of specific binding to an immunoglobulin or T cell receptor. In certain embodiments, the epitope determinant comprises a group of chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl, and in certain embodiments, a specific three-dimensional structural It may have features and / or specific charge features. An epitope is a region of an antigen that is bound by an antibody. In certain embodiments, an antibody is said to specifically bind an antigen upon selective recognition of its target antigen in a complex mixture of proteins and / or macromolecules.

  The term “surface plasmon resonance” as used herein enables real-time analysis of biospecific interactions by detecting changes in protein concentration within the biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For further explanation, see Jonsson, US; (1993) Ann. Biol. Clin. 51: 19-26; Jonsson, U. (1991) Biotechniques 11: 620-627; Johnsson, B. et al. (1995) J. MoI. Mol. Recognit. 8: 125-131; and Johnson, B.M. (1991) Anal. Biochem. 198: 268-277).

The term “k _on ” as used herein refers to an association rate constant for the association of an antibody to an antigen to form an antibody / antigen complex as is known in the art. And

The term “k _off ” as used herein is intended to refer to the dissociation rate constant for dissociation of an antibody from an antibody / antigen complex as is known in the art.

The term “K _d ” as used herein is intended to refer to the dissociation constant of a particular antibody-antigen interaction as is known in the art.

The term “labeled binding protein” as used herein refers to a protein that incorporates a label that allows the identification of the binding protein. Preferably, the label is a detectable marker, such as a radiolabeled amino acid uptake or marked avidin (eg, a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods. Is a linkage of a biotin moiety to a polypeptide that can be detected by streptavidin). Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (eg, ³ H, ¹⁴ C, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹²⁵ I, ¹³¹ I, ¹⁷⁷ Lu, ¹⁶⁶ Ho or ¹⁵³ Sm); fluorescent label (eg, FITC, rhodamine, lanthanide phosphor); enzyme label (eg, horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent marker; biotinyl group; secondary Certain polypeptide epitopes recognized by the reporter (eg, leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags); and magnetic agents such as gadolinium chelators are included.

  The term “antibody complex” refers to a binding protein, such as an antibody, that is chemically linked to a second chemical moiety (such as a therapeutic or cytotoxic agent). In this specification, the term “agent” is used to denote a chemical compound, a mixture of chemical compounds, a biopolymer or an extract prepared from a biological material. Preferably, the therapeutic or cytotoxic agent includes, but is not limited to, pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, Daunorubicin, dihydroxyanthracindione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol and puromycin and analogs or homologs thereof are included.

  The terms “crystal” and “crystallized” as used herein refer to an antibody or antigen-binding portion thereof that exists in crystalline form. Crystals are a form of the solid state of matter, which is different from other forms such as an amorphous solid state or a liquid crystal state. A crystal is composed of a regular repeating three-dimensional array of atoms, ions, molecules (eg, proteins such as antibodies) or molecular assemblies (eg, antigen / antibody complexes). These three-dimensional arrays are arranged according to specific mathematical relationships that are well understood in the art. The basic unit or building block repeated in the crystal is called an asymmetric unit. Repeating asymmetric units in an arrangement that conforms to a predetermined well-ordered crystallographic symmetry results in a “unit cell” of the crystal. Crystals are produced by repeating unit cells with regular translation in all three dimensions. Giege, R.A. And Ducruix, A .; Barrett, Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2nd edition, pp. 197 20 1-16, Oxford University Press, New York, New York (1999).

  As used herein, the term “polynucleotide” refers to a polymeric form of two or more nucleotides (either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide). The term includes single and double stranded forms of DNA, but preferably double stranded DNA.

  As used herein, the term “isolated polynucleotide”, because of its origin, means “isolated polynucleotide” means all or one of the polynucleotides with which the “isolated polynucleotide” is originally found. Is not operably linked to a polynucleotide to which it is not naturally linked; or does not naturally occur as part of a larger sequence (eg, genomic, cDNA or synthetic origin or Means a polynucleotide (of some combination of these).

  As used herein, the term “vector” is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, where additional DNA segments can be linked to the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) can be integrated into the genome of a host cell when introduced into the host cell, thereby replicating with the host genome. In addition, certain vectors are capable of inducing the expression of genes to which they are operably linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply “expression vectors”). In general, expression vectors useful in recombinant DNA technology are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. However, the present invention is intended to include such other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses) that perform equivalent functions.

  The term “operably linked” refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the regulatory sequences. “Operably linked” sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. The term “expression control sequence” as used herein refers to polynucleotide sequences that are necessary to affect the expression and processing of the coding sequences to which they are linked. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that improve translation efficiency (ie, Kozak consensus) Sequences); sequences that enhance the stability of the protein; and optionally, sequences that promote protein secretion. The nature of such regulatory sequences will vary from host organism to host organism, and in prokaryotes such regulatory sequences generally include promoters, ribosome binding sites and transcription termination sequences, and in eukaryotes, such regulatory sequences are generally The sequence can include a promoter and a transcription termination sequence. The term “regulatory sequence” is intended to include components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, eg, leader sequences and fusion partner sequences.

  “Transformation”, as defined herein, refers to any process by which foreign DNA enters a host cell. Transformation can occur under natural or artificial conditions using various methods well known in the art. Transformation can rely on any known method for inserting foreign nucleic acid sequences into prokaryotic or eukaryotic host cells. The method is selected based on the host cell being transformed and can include, but is not limited to, viral infection, electroporation, lipofection and particle bombardment. Such “transformed” cells include stably transformed cells in which the inserted DNA can replicate either as a self-replicating plasmid or as part of a host chromosome. It is. These include cells that transiently express the inserted DNA or RNA for a limited time.

  The term “recombinant host cell” (or simply “host cell”), as used herein, shall refer to a cell into which foreign DNA has been introduced. It should be understood that such terms refer not only to a particular subject cell, but also to the progeny of such a cell. Such progeny may not actually be identical to the parent cell, as certain modifications may occur in subsequent generations, either due to mutations or environmental effects, As used herein, it is still within the scope of the term “host cell”. Preferably, host cells include prokaryotic and eukaryotic cells selected from any sphere of organisms. Preferred eukaryotic cells include protists, fungi, plant and animal cells. Most preferably, host cells include, but are not limited to, prokaryotic cell lines E. coli. Mammalian cell lines CHO, HEK293 and COS; insect cell line Sf9 and fungal cell Saccharomyces cerevisiae.

  Standard techniques can be used for recombinant DNA, oligonucleotide synthesis and tissue culture and transformation (eg, electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly performed in the art or as described herein. In general, the techniques and procedures described above can be performed according to conventional methods well known in the art and as described in various general and specific references cited and discussed throughout this specification. . For example, “Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)” incorporated herein by reference for all purposes). .)checking.

  As is known in the art and as used herein, a “transgenic organism” refers to an organism having cells that contain the transgene, in this case in the organism (or progeny of the organism). The transgene introduced into expresses a polypeptide that is not naturally expressed in the organism. A “transgene” is stably and operably integrated into the genome of a cell from which the transgenic organism develops that controls the expression of the encoded gene product in one or more cell types or tissues of the transgenic organism. DNA construct.

  The terms “regulate” or “modulate” are used interchangeably and as used herein refer to a change or alteration in the activity of a molecule of interest (eg, the biological activity of hRGM A). Modulation can be an increase or decrease in the intensity of certain activities or functions of the molecule of interest. Typical activities and functions of a molecule include, but are not limited to, binding properties, enzyme activity, cell receptor activation and signal transduction.

  Similarly, the term “modulator”, as used herein, is a compound capable of altering or altering the activity or function of a molecule of interest (eg, the biological activity of hRGM A). . For example, a modulator may improve or decrease the strength of certain activities or functions of the molecule as compared to the strength of activity or function observed in the absence of the modulator. As used herein, the term “agonist” refers to a species of molecule as compared to the strength of activity or function observed in the absence of the agonist when contacted with the molecule of interest. Refers to a modulator that improves the strength of activity or function. Specific agonists of interest may include, but are not limited to, hRGM A polypeptide or a polypeptide, nucleic acid, carbohydrate or some other molecule that binds to hRGM A. The term “antagonist” as used herein refers to certain types of molecules compared to the strength of activity or function observed in the absence of the antagonist when contacted with the molecule of interest. Refers to a modulator that reduces the strength of activity or function. Exemplary antagonists include, but are not limited to, proteins, peptides, antibodies, peptibodies, carbohydrates or small organic molecules. Peptibodies are described, for example, in WO 01/83525.

  Specific antagonists of interest include those that block or modulate the biological or immunological activity of hRGM A. Antagonists of hRGM A can include proteins, nucleic acids, carbohydrates or some other molecule that binds to hRGM A, such as, but not limited to, monoclonal antibodies that interact with RGM A molecules. Note that interaction with RGM A can result in binding and neutralization of other ligand / cell membrane components, which can be useful for additive or synergistic functions against multiple diseases.

  As used herein, the term “effective amount” is used to reduce (reduce) or improve the severity and / or duration of a disease or one or more symptoms thereof, Prevent, cause reduction of disease, prevent recurrence, onset, onset or progression of one or more symptoms associated with disease, detect disease or another therapeutic agent (eg, prophylactic or therapeutic agent) The amount of the therapeutic agent that is sufficient to promote or improve the prophylactic or therapeutic effect.

  The term “sample”, as used herein, is used in its broadest sense. A “biological sample” as used herein includes, but is not limited to, any amount of a biologically derived or dead biological material. Such organisms include, but are not limited to, humans, mice, rats, monkeys, dogs, rabbits and other animals. Such materials include, but are not limited to blood, serum, urine, synovial fluid, cells, organs, tissues, bone marrow, lymph nodes and spleen.

2. Polypeptides that bind to hRGM A A major embodiment of the present application comprises an isolated protein or polypeptide that specifically binds to at least one epitope of the RGM A protein. An isolated protein or polypeptide that specifically binds to at least one epitope of the RGM A protein is RGM A against the receptor neogenin of RGM A and / or against bone morphogenetic proteins 2 and 4 (BMP-2, BMP-4) Can be inhibited.

  The most preferred embodiment of the present application comprises an antibody that binds to RGM A or an antigen-binding portion or fragment thereof.

  Preferably, the anti-RGM A antibodies of the invention reduce RGM A activity as assessed, for example, by any one of several in vitro and in vivo assays known in the art or described below. Or high ability to neutralize.

  The present application most preferably neutralizes to RGM A, selectively blocking the binding of RGM A to the receptor of RGM A, to neogenin and to bone morphogenetic proteins 2 and 4 (BMP-2, BMP-4). Including the production of a monoclonal antibody and a neutralizing monoclonal antibody against RGM A that selectively interferes with the binding of RGM A to its co-receptor, bone morphogenetic proteins 2 and 4 (BMP-2, BMP-4).

  Preferably, the monoclonal neutralizing antibody of the present application is a human antibody or a humanized antibody. The term “human antibody” refers to an antibody having variable and constant regions corresponding to or derived from human germline immunoglobulin sequences (see, eg, Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 1991). However, the human antibodies of the present application are amino acid residues that are not encoded by human germline immunoglobulin sequences (eg, by in vitro random or site-directed mutagenesis or in vivo, eg, in CDR, and in particular in CDR3). Mutations introduced by somatic mutation).

  In various embodiments, the antibody is a recombinant antibody or a monoclonal antibody. The most preferred neutralizing antibodies of the present application refer herein to mAb5F9 and mAb8D1 and functional antibody fragments thereof, such as high affinity binding to RGM A with low dissociation rate and high neutralizing ability, such as mAb5F9 and mAb8D1. Other antibodies and functional antibody fragments having equivalent properties to form part of the present invention. The binding affinity and dissociation rate of an anti-RGM A antibody of the present application for an immunogenic RGM A polypeptide or fragment thereof can be determined by any method known in the art. For example, binding affinity can be measured by competitive ELISA, cRIA, BIAcore or KinExA techniques. The dissociation rate can also be measured by BIAcore or KinExA techniques. The binding affinity and dissociation rate are measured by surface plasmon resonance using, for example, BIAcore.

  The mAb5F9 antibody, which is one of the preferred monoclonal antibodies of the present application, comprises a heavy chain variable region (VH region) comprising the sequence of SEQ ID NO: 9 or 34 and a light chain variable region (VL region) comprising the sequence of SEQ ID NO: 10. And at least 90% amino acid sequence identity.

  It is also contemplated that an isolated monoclonal antibody that interacts with RGM A of the present application can be a glycosylated binding protein in which the antibody or antigen-binding portion thereof includes one or more carbohydrate residues. Emerging in vivo protein production can undergo further processing known as post-translational modification. In particular, sugar (glycosyl) residues may be added enzymatically (a process known as glycosylation). The resulting protein with oligosaccharide side chains covalently attached is known as a glycosylated protein or glycoprotein. Protein glycosylation depends on the amino acid sequence of the protein of interest as well as the host cell in which the protein is expressed. The glycosylation enzymes produced by the organism can be different (eg, glycosyltransferases and glycosidases) and the available substrates can be different (nucleotide sugars). Due to such factors, the protein glycosylation pattern and composition of glycosyl residues may vary depending on the host system in which the particular protein is expressed. Glycosyl residues useful in the present invention can include, but are not limited to, glucose, galactose, mannose, fucose, n-acetylglucosamine and sialic acid. Preferably, the glycosylated binding protein comprises a glycosyl residue such that the glycosylation pattern is human.

  The antibodies of the present application comprise heavy chain constant regions such as IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, IgY or IgD constant regions. Furthermore, the antibody can comprise either a light chain constant region, a kappa light chain constant region or a lambda light chain constant region. Preferably, the antibody comprises a kappa light chain constant region. Alternatively, the antibody portion can be, for example, a Fab fragment or a single chain Fv fragment. Substitution of amino acid residues in the Fc portion to alter the function of antibody effectors is known in the art (Winter et al., US Pat. Nos. 5,648,260 and 5,624,821). The Fc portion of an antibody is responsible for several important effector functions such as cytokine induction, ADCC, phagocytosis, complement dependent cytotoxicity (CDC) and half-life / clearance rate of antibodies and antigen-antibody complexes. Mediate. In some cases, these effector functions are preferred for therapeutic antibodies, but in other cases may be unnecessary or harmful depending on the therapeutic purpose. Certain human IgG isotypes, especially IgG1 and IgG3, mediate ADCC and CDC through binding to FcγR and complement C1q, respectively. The neonatal Fc receptor (FcRn) is an important factor that determines the circulating half-life of antibodies. In yet another embodiment, at least one amino acid residue is substituted in the constant region of the antibody, eg, the Fc region of the antibody, such that the function of the antibody effector is altered.

3. Production of anti-hRGM A antibody 3.1. General Principles The antibodies of the present application are suitable for use in suitable hosts (eg, vertebrates including humans, mice, rats, sheep, goats, pigs, cows, horses, reptiles, fish, amphibians and in birds, reptiles and fish eggs). It can be made by immunization. To produce the antibodies of the present application, a host is immunized with an immunogenic RGM A polypeptide of the present invention or a fragment thereof. The term “immunization” as used herein refers to organisms in which the immune repertoire has not been genetically altered or are transgenic (including those modified to present an artificial human immune repertoire). Refers to the process of presenting an antigen to the immune repertoire, whether present or absent. Similarly, an “immunogenic preparation” is a formulation of an antigen containing an adjuvant or other additive that enhances the immunogenicity of the antigen.

  Animals can be immunized by any method known in the art. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Press, 1990. Methods for immunizing non-human animals such as mice, rats, sheep, goats, pigs, cows and horses are well known in the art. See, for example, Harlow and Lane and US Pat. No. 5,994,619. In a preferred embodiment, RGM A antigen is administered with an adjuvant to stimulate an immune response. Such adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptide) or ISCOM (immunostimulatory complex). Such adjuvants can prevent the polypeptide from dispersing rapidly by sequestering the polypeptide for local deposition, or they are chemotactic for macrophages and other components of the immune system. It may contain substances that stimulate the host to secrete factors. Preferably, where a polypeptide is being administered, the immunization schedule includes administration of the polypeptide more than once over a period of weeks.

  It is contemplated that the animal host is immunized with an antigen associated with the cell membrane of intact or broken cells, and the antibodies of the present application are identified by binding to the immunogenic polypeptides of the invention. After immunization of the animal host with the antigen, antibodies can be obtained from the animal. Antibody-containing serum is obtained from the animal by collecting blood or slaughtering the animal. The serum can be used as obtained from the animal, or the immunoglobulin fraction can be recovered from the serum, or the antibody can be purified from the serum. The serum or immunoglobulin obtained in this way is polyclonal and therefore has a different set of properties.

3.2 Anti-RGM A Monoclonal Antibodies Using Hybridoma Technology Monoclonal antibodies can be prepared using a variety of techniques known in the art, including the use of hybridoma, recombinant and phage display technologies or combinations thereof. For example, known in the art, see, for example, Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd edition, 1988); NY (1981), which references are incorporated by reference in their entirety, can be used to generate monoclonal antibodies using hybridoma technology. The term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is made.

  Methods for producing and screening for specific antibodies using hybridoma technology are conventional and well known in the art. In certain embodiments, the invention provides antibodies produced by a method of making a monoclonal antibody and a method comprising culturing a hybridoma cell that secretes an antibody of the invention, wherein preferably the hybridoma is Hybridomas obtained as a result of fusion to spleen cells isolated from a mouse immunized with an antigen of the invention with myeloma cells and then to a hybridoma clone secreting an antibody capable of binding to the polypeptide of the invention It is produced by screening. Briefly, mice can be immunized with RGM A antigen. In preferred embodiments, the antigen is administered with an adjuvant to stimulate an immune response. Such adjuvants include Freund's complete or incomplete adjuvant, RIBI (muramyl dipeptide) or ISCOM (immunostimulatory complex). Such adjuvants can prevent the polypeptide from dispersing rapidly by sequestering the polypeptide for local deposition, or they are chemotactic for macrophages and other components of the immune system. It may contain substances that stimulate the host to secrete certain factors. Preferably, where a polypeptide is being administered, the immunization schedule includes administration of the polypeptide more than once over a period of weeks.

  When an immune reaction is detected, for example, when a specific antibody against the antigen RGM A is detected in mouse serum, the mouse spleen is removed and spleen cells are isolated. The spleen cells are then fused to any suitable myeloma cells (eg, cells from cell line SP20 available from ATCC) by well-known techniques. Hybridomas are selected and cloned by limiting dilution. The hybridoma clones are then assayed by methods known in the art for cells secreting antibodies capable of binding to RGM A. Ascites fluid, which usually contains high levels of antibodies, can be produced by immunizing mice with positive hybridoma clones.

  In another embodiment, antibody-producing immortalized hybridomas can be prepared from immunized animals. After immunization, animals are sacrificed and splenic B cells are fused to immortal myeloma cells as is well known in the art. See, for example, Harlow and Lane, supra. In a preferred embodiment, the myeloma cells do not secrete immunoglobulin polypeptides (non-secreting cell lines). After fusion and antibiotic selection, hybridomas are screened using RGM A or a portion thereof or cells expressing RGM A. In a preferred embodiment, the initial screen is performed using an enzyme linked immunoassay (ELISA) or a radioimmunoassay (RIA) (preferably an ELISA). An example of an ELISA screen is provided in WO00 / 37504, which is incorporated herein by reference.

  As discussed further below, anti-RGM A antibody-producing hybridomas are selected, cloned, and screened for desired characteristics including robust hybridoma growth, high antibody production, and desired antibody characteristics. Hybridomas can be cultured and grown in vivo in syngeneic animals, in immune system deficient animals (eg, nude mice), or in vitro in cell culture. Methods for selecting, cloning and growing hybridomas are well known to those skilled in the art.

  In a preferred embodiment, as described above, the hybridoma is a murine hybridoma. In another preferred embodiment, the hybridoma is produced in a non-human, non-mouse species, such as a rat, sheep, pig, goat, cow or horse. In another embodiment, the hybridoma is a human hybridoma in which a human nonsecretory myeloma is fused with a human cell that expresses an anti-RGM A antibody.

  Antibody fragments that recognize specific epitopes can be generated by known techniques. For example, the Fab of the present invention can be obtained by proteolytic cleavage of an immunoglobulin molecule using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F (ab ′) 2 fragments). F (ab ′) 2 fragments can be generated. The F (ab ') 2 fragment contains the variable region, the light chain constant region and the CH1 domain of the heavy chain.

3.3 Anti-RGM A Monoclonal Antibody Using SLAM In another embodiment of the invention, US Pat. No. 5,627,052, PCT Publication WO 92/02551 and Babcock, J. et al. S. (1996) Proc. Natl. Acad. Sci. The recombinant antibody from single isolated lymphocytes can be obtained using a procedure referred to in the art as the selective lymphocyte antibody method (SLAM), as described in USA 93: 7843-7848. Make it. In this method, an antigen-specific hemolytic plaque assay is used to screen single cells that secrete the antibody of interest (eg, lymphocytes from any one of the immunized animals described above), where: Antigen RGM A, a subunit of RGM A or a fragment thereof is linked to sheep red blood cells using a linker such as biotin to identify a single cell that secretes an antibody with specificity for RGM A used. After identification of the antibody-secreting cells of interest, heavy and light chain variable region cDNAs are removed from the cells by reverse transcriptase-PCR and then the appropriate immunoglobulin constant regions (in mammalian host cells such as COS or CHO cells). These variable regions can be expressed under eg human constant regions). The transfected host cells are then transferred in vitro to the amplified immunoglobulin sequences derived from in vivo selected lymphocytes, eg, by selecting the transfected cells to isolate cells expressing antibodies against RGM A. Further analysis and selection can be made. The amplified immunoglobulin sequences can be further manipulated in vitro, such as by in vitro affinity maturation methods such as those described in PCT Publication WO 97/29131 and PCT Publication WO 00/56772.

3.4 Anti-RGM A Monoclonal Antibodies Using Transgenic Animals In another embodiment of the invention, antibodies are immunized by immunizing non-human animals containing some or all of the human immunoglobulin loci with RGM A antigen. Make it. In a preferred embodiment, the non-human animal is a XENOMOUSE transgenic mouse, a modified mouse line that contains a large fragment of the human immunoglobulin locus and is deficient in mouse antibody production. For example, Green et al., Nature Genetics 7: 13-21 (1994) and US Pat. Nos. 5,916,771, 5,939,598, 5,985,615, 5,998, 209, 6,075,181, 6,091,001, 6,114,598 and 6,130,364. WO91 / 10741 (published on July 25, 1991), WO94 / 02602 (published on February 3, 1994), WO96 / 34096 and WO96 / 33735 (both published on October 31, 1996), WO98 / 16654 (Published on April 23, 1998), WO98 / 24893 (published on June 11, 1998), WO98 / 50433 (published on November 12, 1998), WO99 / 45031 (published on September 10, 1999), WO99 See also / 53049 (published 21 October 1999), WO 00/09560 (published 24 February 2000) and WO 00/037504 (published 29 June 2000). XENOMOUSE transgenic mice produce an adult-like human repertoire of fully human antibodies and produce antigen-specific human Mabs. XENOMOUSE transgenic mice contain approximately 80% of the human antibody repertoire through the introduction of megabase-sized germline structural YAC fragments at the human heavy and x light chain loci. Mendez et al., Nature Genetics 15: 146-156 (1997), Green and Jakobovits J. et al. Exp. Med. 188: 483-495 (1998), the disclosures of which are incorporated herein by reference.

3.5 Anti-RGM A Monoclonal Antibodies Using a Recombinant Antibody Library In vitro methods can also be used to make the antibodies of the invention, in which case to identify antibodies with the desired binding specificity: Screen antibody libraries. Methods for such screening of recombinant antibody libraries are well known in the art and include, for example, Ladner et al., US Pat. No. 5,223,409; Kang et al., PCT Publication WO 92/18619; Dower et al., PCT publication WO 91/17271; Winter et al., PCT publication WO 92/20791; Markland et al., PCT publication WO 92/15679; Breitling et al., PCT publication WO 93/01288; McCafferty et al., PCT publication WO 92/01047; Garrard et al., PCT publication. WO92 / 09690; Fuchs et al. (1991), Bio / Technology 9: 1370-1372; Hay et al. (1992) Hum Antibody Hybridomas 3: 81-85; Hus e et al. (1989), Science 246: 1275-1281; McCafferty et al., Nature (1990) 348: 552-554; Griffiths et al. (1993) EMBO J 12: 725-734; Hawkins et al. (1992) J Mol Biol 226: 889. Clackson et al. (1991) Nature 352: 624-628; Gram et al. (1992) PNAS 89: 3576-3580; Garrad et al. (1991) Bio / Technology 9: 1373-1377; Hoogenboom et al. (1991) Nuc Acid Res. : 4133-4137; and Barbas et al. (1991) PNAS 88: 7978-7982, US Application Publication No. 20030186374 and PCT Publication. The methods described in WO 97/29131, the contents of each of which are incorporated herein by reference, are included.

The recombinant antibody library can be from a subject immunized with RGM A or a portion of RGM A. Alternatively, the recombinant antibody library can be from an untreated subject, ie, a subject that has not been immunized with RGM A, such as a human antibody library from a human subject that has not been immunized with human RGM A. The antibodies of the invention are selected by screening a recombinant antibody library with peptides containing human RGM A, thereby selecting antibodies that recognize RGM A. Methods for performing such screening and selection are well known in the art, such as those described in the references in the preceding paragraph. To select antibodies of the invention having a specific binding affinity for RGM A, such as those that dissociate from human RGM A with a specific k _off rate constant, methods known in the art of surface plasmon resonance are used. Thus, an antibody having a desired k _off rate constant can be selected. Use standard methods known in the art to assess inhibition of hRGM A activity to select antibodies of the invention having specific neutralizing activity against RGM A, such as those with specific IC ₅₀ Can do.

  In certain embodiments, the present invention relates to an isolated antibody or antigen-binding portion thereof that binds to human RGM A. Preferably, the antibody is a neutralizing antibody. In various embodiments, the antibody is a recombinant antibody or a monoclonal antibody.

  For example, the antibodies of the present invention can also be generated using various phage display methods known in the art. In the phage display method, a functional antibody domain is displayed on the surface of a phage particle bearing a polynucleotide sequence encoding the functional antibody domain. In particular, such phage can be used to display an antigen binding domain expressed from a repertoire or combinatorial antibody library (eg, human or mouse). Antigens can be used to select or identify phage that express an antigen binding domain that binds to the antigen of interest, for example, using labeled antigen or antigen bound or captured to a solid surface or bead. The phage used in these methods usually have fd and M13 binding domains expressed from the phage along with Fab, Fv or disulfide stabilized Fv antibody domains that are recombinantly fused to either phage gene III or gene VIII protein. It is a linear phage containing. Examples of phage display methods that can be used to make the antibodies of the present invention include Brinkman et al. Immunol. Methods 182: 41-50 (1995); Ames et al., J. Biol. Immunol. Methods 184: 177-186 (1995); Kettleborough et al., Eur. J. et al. Immunol. 24: 952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57: 191-280 (1994); PCT / GB91 / 01134; PCT publication WO 90/02809; WO 91 / 10737; WO92 / 01047; WO92 / 18619; WO93 / 11236; WO95 / 15982; WO95 / 20401; and US Pat. Nos. 5,698,426; 5,223,409; 5,403,484. No. 5,580,717; No. 5,427,908; No. 5,750,753; No. 5,821,047; No. 5,571,698; 427,908; 5,516,637; 5,780,225 No. 5,658,727; Nos. 5,733,743 and 5,969,108, each of which is incorporated herein by reference in its entirety. Is included.

As described in the above references, for example, as described in detail below, any desired host (mammalian cells, insect cells, plant cells, yeasts and bacteria, including human antibodies or any other desired antigen-binding fragment) After phage selection, the phage-derived antibody coding region can be isolated and used to generate the whole antibody expressed in. See, e.g., PCT Publication WO 92/22324; Mullinax et al., BioTechniques 12 (6): 864-869 (1992); and Sawai et al., AJRI 34: 26-34 (1995); and Better et al., Science 240: 1041-1043 (1988). (These references are incorporated by reference in their entirety.) Using methods known in the art, such as those disclosed in, Fab, Fab ′ and F (ab ′) ₂ fragments Techniques for recombinant production can also be used. Examples of techniques that can be used to generate single chain Fv and antibodies include US Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203: 46. -88 (1991); Shu et al., PNAS 90: 7995-7999 (1993); and Skerra et al., Science 240: 1038-1040 (1988).

  Instead of screening recombinant antibody libraries by phage display, other methods known in the art for screening large combinatorial libraries can be applied to the identification of bispecific antibodies of the invention. One type of alternative expression system is PCT publication WO 98/31700 by Szostak and Roberts and Roberts, R.A. W. And Szostak, J. et al. W. (1997) Proc. Natl. Acad. Sci. USA 94: 12297-12302, where a recombinant antibody library is expressed as an RNA-protein fusion. In this system, in vitro translation of a synthetic mRNA having puromycin, a peptidyl acceptor antibiotic at its 3 'end results in a covalent fusion between the mRNA and the peptide or protein that it encodes. Thus, a complex mixture of mRNA (eg, combinatorial library) based on the characteristics of the encoded peptide or protein, eg, an antibody or part thereof (such as binding of the antibody or part thereof to a bispecific antigen). Thus, specific mRNA can be concentrated. Nucleic acid sequences encoding antibodies or portions thereof recovered from such library screening can be expressed by recombinant means as described above (eg, in mammalian host cells), and further as described above, Either by a further round of screening for mRNA-peptide fusions in which the mutation has been introduced into the originally selected sequence, or by other methods for in vitro affinity maturation of recombinant antibodies, It can be subjected to further affinity maturation.

  In another approach, the antibodies of the present invention can also be generated using yeast display methods known in the art. In yeast display methods, genetic methods are used to tether antibody domains to the yeast cell wall and present them on the surface of the yeast. In particular, such yeast can be used to display antigen binding domains expressed from a repertoire or combinatorial antibody library (eg, human or mouse). Examples of yeast display methods that can be used to make the antibodies of the invention are disclosed in Wittrup et al., US Pat. No. 6,699,658, which is incorporated herein by reference. Things are included.

4). Production of Specific Recombinant RGM A Antibodies of the Invention Antibodies of the invention can be produced by any of a number of techniques known in the art. For example, expression from a host cell, in which case expression vectors encoding heavy and light chains are transfected into the host cell by standard techniques. Various forms of the term “gene transfection” are intended to encompass a wide variety of techniques commonly used for the introduction of foreign DNA into prokaryotic or eukaryotic host cells (eg, electroporation). , Calcium phosphate precipitation, DEAE-dextran gene transfer, etc.). While it is possible to express the antibodies of the invention in either prokaryotic or eukaryotic host cells, eukaryotic cells (and particularly mammalian cells) are more correctly folded and immunologically active than prokaryotic cells. Expression of antibodies in eukaryotic cells is preferred, and expression in mammalian host cells is most preferred because it is likely to construct and secrete antibodies.

  Preferred mammalian host cells for expressing recombinant antibodies of the invention include Chinese hamster ovary (CHO cells) (eg RJ Kaufman and PA Sharp (1982) Mol. Biol. 159: 601-621). And dhfr-CHO cells as described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77: 4216-4220, as used in conjunction with a DHFR selectable marker, as described in Cells, COS cells and SP2 cells are included. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, it allows expression of the antibody in the host cell or, more preferably, allows secretion of the antibody into the medium in which the host cell is grown. Antibodies are produced by culturing host cells for a time sufficient to do so. Antibodies can be recovered from the culture medium using standard protein purification methods.

  Host cells can also be used to generate functional antibody fragments such as Fab fragments or scFv molecules. It should be understood that variations in the above procedure are within the scope of the present invention. For example, it may be desirable to introgress DNA encoding a functional fragment of either the light chain and / or heavy chain of an antibody of the invention into a host cell. Recombinant DNA technology can also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that are not required for binding to the antigen of interest. Molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the present invention. Furthermore, by cross-linking the antibody of the present invention to the second antibody by standard chemical cross-linking methods, one heavy chain and one light chain are antibodies of the present invention and the other heavy chain and light chain are Bifunctional antibodies can be made that are specific for antigens other than the antigen of interest.

  In a preferred system for recombinant expression of an antibody of the invention or antigen-binding portion thereof, a recombinant expression vector encoding both antibody heavy and light chains is introduced into dhfr-CHO cells by calcium phosphate-mediated gene transfer. . Within the recombinant expression vector, the antibody heavy and light chain genes are operably linked to CMV enhancer / AdMLP promoter regulatory elements, respectively, to drive high level transcription of the gene. The recombinant expression vector also has a DHFR gene, which allows selection of CHO cells into which the vector has been transfected using methotrexate selection / amplification. To allow expression of the antibody heavy and light chains, the selected transformed host cells are cultured and intact antibody is recovered from the medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfer to the host cell, select for transformants, culture the host cell, and recover the antibody from the culture medium. Furthermore, the present invention provides a method for synthesizing the recombinant antibody of the present invention by culturing the host cell of the present invention in an appropriate medium until the recombinant antibody of the present invention is synthesized. The method further includes isolating the recombinant antibody from the medium.

4.1 Anti-RGM A antibodies Table 5 lists the amino acid sequences of the VH and VL regions of preferred anti-hRGM A antibodies of the invention.

  The previously described isolated anti-RGM A antibody CDR sequences establish a novel family of RGM A binding proteins, including polypeptides comprising CDR sequences, isolated according to the present invention. To make and select CDRs of the invention that have preferred RGM A binding and / or neutralize activity with respect to hRGM A, but not limited to, those specifically described herein. Standard methods known in the art can be used to make the binding proteins of the invention, including and assess RGM A binding and / or neutralization of the properties of these binding proteins.

4.2 Anti-RGM A chimeric antibody A chimeric antibody is a molecule in which the animal species derived from the region of the antibody differs, such as an antibody having a variable region derived from a mouse monoclonal antibody and a human immunoglobulin constant region. Methods for making chimeric antibodies are known in the art. See, for example, Morrison, Science 229: 1202 (1985); Oi et al., BioTechniques 4: 214 (1986); Gillies et al. (1989) J. MoI. Immunol. Methods 125: 191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are hereby incorporated by reference in their entirety. See). Furthermore, techniques developed for the production of “chimeric antibodies” by splicing genes from appropriate antigen-specific mouse antibody molecules together with genes from appropriate biologically active human antibody molecules (Morrison et al., 1984). Proc. Natl. Acad. Sci. 81: 851-855; Neuberger et al., 1984, Nature 312: 604-608; Takeda et al., 1985, Nature 314: 452-454, which are hereby incorporated by reference in their entirety. Can be used).

  In certain embodiments, chimeric antibodies of the invention are made by replacing the heavy chain constant region of a mouse monoclonal anti-human RGM A antibody described herein with a human IgG1 constant region.

4.3 Anti-RGM A CDR-grafted antibody The CDR-grafted antibody of the present invention has one or more of the CDR regions of _VH and / or _VL replaced with a non-human CDR sequence such as a mouse antibody of the present invention. Contains heavy and light chain variable region sequences from human antibodies. Framework sequences from any human antibody can serve as a template for CDR grafting. However, such linear substitution on the framework often results in some loss of binding affinity for the antigen. The more homologous a human antibody is to the original mouse antibody, the less likely it is to introduce distortion in the CDR that can reduce affinity by combining the mouse CDR with the human framework. Accordingly, it is preferred that the human variable framework selected to replace the mouse variable framework excluding the CDRs has at least 65% sequence identity with the mouse antibody variable region framework. More preferably, the human and mouse variable regions excluding the CDRs have at least 70% sequence identity. Even more preferably, the human and mouse variable regions, excluding the CDRs, have at least 75% sequence identity. Most preferably, the human and mouse variable regions, excluding the CDRs, have at least 80% sequence identity. Methods for making CDR grafted antibodies are known in the art (Jones et al., Nature 321: 522-525 (1986); US Pat. No. 5,225,539). In a specific embodiment, the present invention provides CDR grafted antibodies having V _H and / or V _L chains as described in Table 6.

4.4 Anti-RGM A Humanized Antibody A humanized antibody is a non-human species that binds to a desired antigen having one or more complementarity determining regions (CDRs) from non-human species and framework regions from human immunoglobulin molecules. It is an antibody molecule derived from an antibody. Known human Ig sequences are described, for example, at www. ncbi. nlm. nih. gov / entrez- / query. fcgi; www. atcc. org / phage / hdb. html; www. scquest. com /; www. abcam. com /; www. antibodyresource. com / online comp. html; www. public. istate. edu /. about. pedro / research_tools. html; www. megn. uni-heidelberg. de / SD / IT / IT. html; www. whfreeman. com / immunology / CH-05 / kuby05. html; www. library. thinkquest. org / 12429 / Immune / antibody. html; www. hhmi. org / grants / lectures / 1996 / vlab /; www. path. cam. ac. uk /. about. mrc7 / m-ikeimages. html; www. antibodyresource. com /; mcb. harvard. edu / BioLinks / Immunology. html. www. immunologiclink. com /; pathbox. Wustl. edu /. about. hcenter / index. -Html; www. biotech. ufl. edu /. about. hcl /; www. pebio. com / pa / 340913/340913. html-; www. nal. usda. gov / awic / pubs / antibody /; www. m. ehime-u. acjp /. about. yasuhito- / Elisa. html; www. biodesign. com / table. asp; www. icnet. uk / axp / facs / davies / lin-ks. html; www. biotech. ufl. edu /. about. fccl / protocol. html; www. isac-net. org / sites_geo. html; Aximl. imt. uni-marburg. de /. about. lek / AEP-Start. html; uci. kun. nl /. about. jraats / linksl. html; www. recab. uni-hd. de / immuno. bme. nwu. edu /; www. mrc-cpe. cam. ac. uk / imt-doc / pu-blic / INTRO. html; www. ibt. unam. mx / vir / V_mice. html; imgt. cnusc. fr: 8104 /; www. biochem. ucl. ac. uk /. about. martin / abs / index. html; antibody. bath. ac. uk /; abgen. cvm. tamu. edu / lab / wwwwagen. html; www. unith. ch /. about. honegger / AHOseminar / Slide01. html; www. cryst. bbk. ac. uk /. about. ubcg07s /; www. nimr. mrc. ac. uk / CC / ccaewg / ccaewg. html; www. path. cam. ac. uk /. about. mrc7 / h-umanation / TAHHP. html; www. ibt. unam. mx / vir / structure / stat_aim. html; www. biosci. misouri. edu / smithgp / index. html; www. cryst. bioc. cam. ac. uk /. about. fmolina / Web-pages / Pept / spottech. html; www. jerini. de / fr products. html; www. patents. ibm. com / ibm. html. Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Pat. S. Dept. Health (1983), each incorporated herein by reference in its entirety. Reduce immunogenicity, or reduce, promote or change binding, affinity, association rate, dissociation rate, binding power, specificity, half-life or some other suitable property as known in the art In order to do this, such imported sequences can be used.

  Framework residues in the human framework regions can be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions can be performed by methods well known in the art, for example, modeling of CDR and framework residue interactions to identify framework residues important for antigen binding and antigen binding and specific positions. Identified by sequence comparisons to identify unusual framework residues at. (See, eg, Queen et al., US Pat. No. 5,585,089; Riechmann et al., Nature 332: 323 (1988), which are incorporated herein by reference in their entirety). Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs that show and present putative three-dimensional conformational structures of selected candidate immunoglobulin sequences are available. Examination of these presentations allows analysis of the possible role of residues in the function of a candidate immunoglobulin sequence, ie, analysis of residues that affect the ability of a candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the consensus and import sequences so that the desired antibody characteristic, such as improved affinity for the target antigen (s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding. Antibodies include, but are not limited to, Jones et al., Nature 321: 522 (1986); Verhoeyen et al., Science 239: 1534 (1988)), Sims et al. Immunol. 151: 2296 (1993); Chothia and Lesk, J. MoI. Mol. Biol. 196: 901 (1987), Carter et al., Proc. Natl. Acad. Sci. U. S. A. 89: 4285 (1992); Presta et al., J. Biol. Immunol. 151: 2623 (1993), Padlan, Molecular Immunology 28 (4/5): 489-498 (1991); Studnikka et al., Protein Engineering 7 (6): 805-814 (1994); 973 (1994); PCT Publication WO91 / 09967, PCT /: US98 / 16280, US96 / 18978, US91 / 09630, US91 / 05939, US94 / 01234, GB89 / 01334, GB91 / 01134, GB92 / 01755; WO90 / 14443, WO90 / 14424, WO90 / 14430, EP229,246, EP592,106; EP519,596, EP239,400, US Pat. No. 5, 565,332, 5,723,323, 5,976,862, 5,824,514, 5,817,483, 5,814,476, 5,763,192, 5,723,323, 5,766,886, 5,714,352, 6,204,023, 6,180 , 370, 5,693,762, 5,530,101, 5,585,089, 5,225,539; 4,816,567 (respectively Which are generally incorporated herein by reference, including references cited therein), and can be humanized using various techniques known in the art.

5. Further Embodiments of Antibodies of the Invention 5.1 Fusion Antibodies and Immunoadhesions This application also includes fusion antibodies or immunoadhesions that can be made to include all or part of the RGM A antibodies of the present application linked to another polypeptide. Also described. In certain embodiments, only the variable region of the RGM A antibody is linked to the polypeptide. In other embodiments, the VH domain of the RGM A antibody of the present application is linked to a first polypeptide, while the VL domain of this antibody allows the VH and VL domains to interact with each other to form an antibody binding site. To a second polypeptide that binds to the first polypeptide. In other embodiments, the VH domain is separated from the VL domain by a linker that allows the VH and VL domains to interact with each other (see single chain antibodies below). The VH-linker-VL antibody is then linked to the polypeptide of interest. This fusion antibody is useful for directing the polypeptide to a cell or tissue expressing RGMA. The polypeptide of interest can be a therapeutic agent such as a toxin, or can be a diagnostic agent such as an enzyme that can be easily visualized, such as horseradish peroxidase. In addition, fusion antibodies can be made such that two (or more) single chain antibodies are linked together. This is useful when it is desired to produce a bivalent or multivalent antibody on a single polypeptide chain or when a bispecific antibody is desired to be produced.

  Certain embodiments provide a labeled binding protein wherein an antibody or antibody portion of the present application is derivatized or linked to another functional molecule (eg, another peptide or protein). For example, to one or more other molecular moieties capable of mediating the association of an antibody or antibody moiety with another molecule (such as a streptavidin core region or a polyhistidine tag), eg, a nucleic acid, another antibody (eg, Bifunctional antibodies or diabodies), detectable reagents, cytotoxic agents, pharmaceuticals and / or proteins or peptides by functionally linking the antibody or antibody portion of the present application (chemical coupling, gene fusion, non-covalent) The labeled binding protein of the present application can be derivatized by binding or otherwise.

  Useful detectable reagents with which the antibodies or antibody portions of the present application can be derivatized include fluorescent compounds. Exemplary fluorescent detectable reagents include fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, and the like. The antibody can also be derivatized with a detectable enzyme such as alkaline phosphatase, horseradish peroxidase, glucose oxidase. If the antibody is derivatized with a detectable enzyme, this is detected by adding additional reagents that the enzyme uses to produce a detectable reaction product. For example, in the presence of the detectable reagent horseradish peroxidase, the addition of hydrogen peroxide and diaminobenzidine results in a colored reaction product that is detectable. Antibodies can also be derivatized with nucleic acids and biotin and detected through indirect measurement of avidin or streptavidin binding.

5.2 Single chain antibodies This application includes single chain antibodies (scFv) that bind to the immunogenic RGM A of the present invention. To generate scFv, DNA encoding VH- and V encodes a flexible linker such that the VH and VL sequences can be expressed as a continuous single chain protein in which the VL and VH regions are linked by a flexible linker. For example, operably linked to DNA encoding the amino acid sequence (Gly-4-Ser) (see, eg, Bird et al. (1988) Science 242: 423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883; McCafferty et al., 30 Nature (1990) 34 8: 552-554). Single chain antibodies can be monovalent when only one VH and VL are used, bivalent when two VH and VL are used, or three or more VH and VL are used. If it is, it is multivalent. Two of these scFv fragments linked via a linker are called “diabodies” and this form is also encompassed by the present invention.

5.3 Bispecific Antibodies The present application further includes bispecific antibodies or antigen-binding fragments thereof, wherein one specificity is for the immunogenic RGM A polypeptide of the present application. For example, bispecific antibodies can be made that specifically bind to an immunogenic RGM A polypeptide of the invention through one binding domain and to a second molecule through a second binding domain. In addition, a plurality of VHs and VLs that specifically bind to another molecule involved in attenuating myelin-mediated growth cone collapse and inhibiting neurite outgrowth and sprouting into the immunogenic polypeptides of the invention. Containing single chain antibodies can be made. Such bispecific antibodies can be made using well known techniques such as, for example, Fanger et al., Immuno Methods 4: 72-81 (1994) and Wright and Harris, 20 (supra).

  In certain embodiments, bispecific antibodies are prepared using one or more of the variable regions derived from an antibody of the invention. In another embodiment, bispecific antibodies are prepared using one or more CDR regions from said antibody.

5.4 Derivatized and labeled antibodies The antibodies or antigen-binding fragments of the present application are derivatized or linked to another molecule (eg, another peptide or protein). In general, the antibody or antigen-binding fragment is derivatized such that binding to an immunogenic polypeptide of the invention is not adversely affected by derivatization or labeling.

For example, another antibody (e.g., one that can mediate association of an antibody or antibody-binding fragment with one or more other molecular moieties, e. Bispecific antibodies or diabodies), detection reagents, cytotoxic drugs, pharmaceuticals and / or proteins or peptides functionally (by chemical coupling, gene fusion, non-covalent or otherwise) Can be linked. Still further, the antibody or antigen-binding portion thereof can be part of a larger immunoadhesion molecule formed by covalent or non-covalent binding of the antibody or antibody portion with one or more other or different proteins or peptides. Examples of such immunoadhesion molecules include the use of the streptavidin core region to generate tetrameric scFv molecules (Kiprianonov et al. (1995) Human Antibodies and Hybridomas 6: 93-101) and divalent and biotinylated scFv Use of cysteine residues, marker peptides and C-terminal polyhistidine tags (Kipriyanov et al. (1994) Molecular Immunology 31: 1047-1058) to make the molecule is included. Some of the antibodies, such as Fab and F (ab ′) ₂ fragments, can be prepared from the whole antibody using conventional techniques such as papain or pepsin digestion of the whole antibody, respectively. In addition, standard recombinant DNA techniques can be used to obtain antibodies, antibody portions, and immunoadhesion molecules.

  Derivatized antibodies can be made by cross-linking two or more antibodies (of the same type or of different types, eg, to make bispecific antibodies). Suitable crosslinkers include those that are heterobifunctional (eg m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homofunctional with two different reactive groups separated by a suitable spacer (Eg disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, Ill.

  The derivatized antibody can also be a labeled antibody. For example, detection reagents to which the antibodies or antibody portions of the present invention can be derivatized are fluorescent compounds including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the like. is there. The antibody can also be labeled with enzymes that are useful for detecting horseradish peroxidase, galactosidase, luciferase, alkaline phosphatase, glucose oxidase, and the like. In embodiments labeled with a detectable enzyme, the antibody is detected by adding additional reagents that the enzyme uses to produce a detectable reaction product. For example, hydrogen peroxide and diaminobenzidine and horseradish peroxidase. The antibody can also be labeled with biotin and detected through indirect measurement of avidin or streptavidin binding. The antibody can also be labeled with a predetermined polypeptide epitope recognized by the secondary reporter (eg, leucine zipper pair sequence, binding site for the secondary antibody, metal binding domain, epitope: tag). The RGM A antibody or antigen fragment thereof can also be labeled with a radiolabeled amino acid. This radioactive label can be used for both diagnostic and therapeutic purposes. A radiolabeled RGM A antibody can be used diagnostically, for example, to determine RGM A receptor levels in a subject. Furthermore, radiolabeled RGM A antibodies can be used therapeutically to treat spinal cord injury.

Examples of labels for polypeptides include, but are not limited to, the following radioisotopes or radionucleotides: ¹⁵ N, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹²⁵ I, ¹³¹ I, ¹⁷⁷ Lu, ¹⁶⁶ Ho ¹⁵³ Sm. RGM A antibodies or antigenic fragments thereof can also be derivatized with chemical groups such as polyethylene glycol (PEG), methyl or ethyl groups or carbohydrate groups. These groups can be useful to improve the biological properties of the antibody, for example, to increase serum half-life or to increase tissue binding. In addition, the label for the polypeptide can include nucleic acids, for example, siRNA for suppressing gene expression in cells or tissues having DNA or RGM A for detection by PCR or for promoting gene expression.

  The class and subclass of RGM A antibodies can be determined by any method known in the art. In general, the class and subclass of an antibody can be determined using antibodies that are specific for a particular class and subclass of antibody. Such antibodies are commercially available. The class and subclass can be determined by ELISA, Western blot and other techniques. Alternatively, classes and subclasses sequence all or part of the constant domain of an antibody heavy and / or light chain and compare their amino acid sequences with known amino acid sequences of various classes and subclasses of immunoglobulins And can be determined by determining the class and subclass of the antibody.

5.5 Dual Variable Domain Immunoglobulin A dual variable domain (DVD) binding protein or immunoglobulin, as used herein, is a binding protein that includes two or more antigen binding sites, eg, bivalent and It is a multivalent binding protein such as tetravalent. The term “multivalent binding protein” is used herein to denote a binding protein comprising two or more antigen binding sites. The multivalent binding protein is preferably modified to have more than one antigen binding site and is usually not a natural antibody. The term “multispecific binding protein” refers to a binding protein capable of binding to two or more related or unrelated targets. Such DVDs can be monospecific, i.e. capable of binding to one antigen, or multispecific, i.e., capable of binding to more than one antigen. A DVD binding protein comprising two heavy chain DVD polypeptides and two light chain DVD polypeptides refers to DVD Ig. Each half of a DVD Ig contains a heavy chain DVD polypeptide and a light chain DVD polypeptide and two antigen binding sites. Each binding site includes a heavy chain variable domain and a light chain variable domain with a total of 6 CDRs involved in antigen binding for each antigen binding site. DVD binding proteins and methods of making DVD binding proteins are disclosed in US patent application Ser. No. 11 / 507,050 (incorporated herein by reference). The present invention is intended to include a DVD binding protein comprising a binding protein capable of binding to RGM A. Preferably, the DVD binding protein is capable of binding to RGM A and a second target. The second target is anti-inflammatory MAB activity (IL-1, IL-6, IL-8, IL-11, IL-12, IL-17, IL-18, IL-23, TNFα / β, IFN- Transport mediation from β, γ, LIF, OSM, CNTF, PF-4, platelet basic protein (PBP), NAP-2, β-TG, MIP-1, MCP2 / 3, RANTES, lymphotactin) From proteins (insulin receptor, transferrin receptor, thrombin receptor, leptin receptor, LDL receptor), other nerve regeneration MAB (NgR, Lingo, p75, CSPG (eg, NG-2, neurocan, brevican, versican, Aggrecan) hyaluronic acid, mAG, tenacin, NI-35, NI-250, IMP, perleca , Neurocan, phosphacan, nogo-A, OMGP, Sema4D, Sema3A, ephrinB3, ephrinA2, ephrinA5, MAG, EphA4, plexinB1, TROY, wnts, ryk rec., BMP-2, BMP-4, BMP- 7) From neuroprotective MAB activity (EGF, EGFR, Sema3), from anti-amyloid βMAB (eg m266, 3D6 (vapineuzumab), anti-spheroid MAB 7C6), from CNS localized receptors and transporters (serotonin receptors) Body, dopamine receptor, DAT, Asc-1, GlyT1).

5.6 Bispecific Antibodies This application also describes “bispecific antibody” technology. Bispecific antibodies can act as agonists, antagonists, or both in various combinations. A bispecific antibody is an antibody in which a VH chain binds to a first antigen and a VL chain binds to another antigen, as exemplified in WO2008082651.

5.7 Crystallized Antibody Another embodiment of the present application provides a crystallized binding protein. The term “crystallization” as used herein refers to an antibody or antigen-binding portion thereof that exists in crystalline form. Crystals are one form of the solid state of the substance and are different from other forms such as an amorphous solid state or a liquid crystal state. Crystals are composed of regularly repeated three-dimensional arrays of atoms, ions, molecules (eg, proteins such as antibodies) or molecular assemblies (eg, antigen / antibody complexes). These three-dimensional arrays are arranged according to specific mathematical relationships that are well understood in the art. The basic unit or building block repeated in the crystal is called an asymmetric unit. Repeating asymmetric units in an arrangement that conforms to a predetermined well-ordered crystallographic symmetry results in a “unit cell” of the crystal. Crystals are produced by repeating unit cells with regular translation in all three dimensions. Giege, R.A. And Ducruix, A .; Barrett, Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2nd edition, pp. 199 20 1-16, Oxford University Press, New York, New York (1999).

  Preferably, this application describes crystals of all RGM A antibodies and fragments thereof as disclosed herein, and formulations and compositions comprising such crystals. In certain embodiments, the crystallized binding protein has a longer half-life in vivo than the soluble form of the binding protein. In another embodiment, the binding protein retains biological activity after crystallization.

  The crystallized binding proteins of the present invention can be made according to methods known in the art and as disclosed in International Application Publication No. WO 02/072636, which is incorporated herein by reference.

5.8 Glycosylated antibodies Another embodiment of the invention provides glycosylated binding proteins wherein the antibody or antigen binding portion thereof comprises one or more carbohydrate residues. Emerging in vivo protein production can undergo further processing known as post-translational modification. In particular, sugar (glycosyl) residues can be added enzymatically, a process known as glycosylation. The resulting protein with oligosaccharide side chains covalently attached is known as a glycosylated protein or glycoprotein. An antibody is a glycoprotein having one or more carbohydrate residues in the Fc domain as well as the variable domain. The carbohydrate residues of the Fc domain have an important effect on the effector function of the Fc domain, with minimal effect on the antigen binding or half-life of the antibody (R. Jefferis, Biotechnol. Prog. 21 (2005), pp. 11-16). On the other hand, glycosylation at the variable domain can affect the antigen-binding activity of the antibody. Can variable glycosylation have a negative effect on antibody binding affinity, probably due to steric hindrance (Co, MS et al., Mol. Immunol. (1993) 30: 1361-1367), or As a result, the affinity for the antigen can be improved (Wallick, SC et al., Exp. Med. (1988) 168: 1099-1109; Wright, A. et al., EMBO J. (1991) 10: 2717-2723). ).

  One aspect of the invention relates to making glycosylation site mutants in which the O- or N-linked glycosylation sites of the binding protein are mutated. One skilled in the art can make such mutants using standard well-known techniques. Glycosylation site mutants that retain biological activity but have increased or decreased binding activity are another object of the present invention.

  In yet another embodiment, glycosylation of the antibody or antigen binding portion of the invention is modified. For example, an unglycosylated antibody can be made (ie, the antibody lacks glycosylation). For example, glycosylation can be altered to improve the affinity of the antibody for the antigen. Such sugar modifications can be made, for example, by changing one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in removal of one or more variable region glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation can improve the affinity of the antibody for the antigen. Such an approach is further described in PCT Publication WO2003016466A2 and US Pat. Nos. 5,714,350 and 6,350,861, each of which is incorporated herein by reference in its entirety. It is described in detail.

  Additionally or alternatively, modified antibodies of the invention with altered glycosylation types can be made, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased branched GlcNAc structures. Such modified glycosylation patterns have been shown to improve the ADCC ability of antibodies. For example, such sugar modifications can be made by expressing the antibody in host cells with altered glycosylation mechanisms. Cells with altered glycosylation mechanisms have been described in the art and can be used as host cells to express the recombinant antibodies of the invention, thereby producing antibodies with altered glycosylation. See, for example, Shields, R.A. L. (2002) J. et al. Biol. Chem. 277: 26733-26740; Umana et al. (1999) Nat. Biotech. 17: 176-1 and European Patent No. EP 1,176,195; PCT Publication Nos. WO 03/035835 and WO 99/5434280, each of which is incorporated herein by reference in its entirety.

  Protein glycosylation depends on the amino acid sequence of the protein of interest as well as the host cell in which the protein is expressed. Different organisms can have different glycosylation enzymes (eg, glycosyltransferases and glycosidases) and available substrates (nucleotide sugars). Due to such factors, the protein glycosylation pattern and composition of glycosyl residues may vary depending on the host system in which the particular protein is expressed. Glycosyl residues useful in the present invention can include, but are not limited to, glucose, galactose, mannose, fucose, n-acetylglucosamine and sialic acid. Preferably the glycosylated binding protein comprises a glycosyl residue such that the glycosylation pattern is human.

  It is known to those skilled in the art that protein characteristics can differ as a result of various protein glycosylation. For example, the efficiency of a therapeutic protein produced in a microbial host such as yeast and glycosylated using the yeast endogenous pathway is compared to the efficiency of the same protein expressed in a mammalian cell such as a CHO cell line. May have been reduced. Such glycoproteins can also be immunogenic in humans and have a short in vivo half-life after administration. Specific receptors in humans and other animals can recognize specific glycosyl residues and promote rapid clearance of proteins from the bloodstream. Other deleterious effects may include protein folding, solubility, sensitivity to proteases, trafficking, transport, compartmentalization, secretion, recognition by other proteins or factors, antigenicity or allergenic changes. Thus, the practitioner will have a glycosylation composition and pattern that is identical or at least similar to that produced in human cells or species-specific cells of the intended subject animal. You may like the therapeutic protein you have.

  Expression of glycosylated proteins different from that of the host cell can be achieved by genetically modifying the host cell to express a heterologous glycosylation enzyme. Using techniques known in the art, practitioners can generate antibodies or antigen-binding portions thereof that exhibit human protein glycosylation. For example, yeast strains express non-natural glycosylation enzymes such that the glycosylated proteins (glycoproteins) produced in these yeast strains exhibit the same protein glycosylation as that of animal cells, particularly human cells. (US Patent Application Publications 20040018590 and 200201337134 and PCT Publication WO2005 / 100584A2).

  Furthermore, it will be appreciated by those skilled in the art that host cells that have been engineered to express various glycosylation enzymes such that the member host cells of the library produce a protein of interest having a variant glycosylation pattern. The library can be used to express the protein of interest. The practitioner can then select and isolate the protein of interest having a particular novel glycosylation pattern. Preferably, proteins with a particularly selected novel glycosylation pattern exhibit an improvement or change in biological properties.

5.9 Anti-idiotype antibodies In addition to binding proteins, the invention also relates to anti-idiotype (anti-Id) antibodies specific for such binding proteins of the invention. An anti-Id antibody is an antibody that recognizes a unique determinant that generally associates with the antigen-binding region of another antibody. Anti-Id can be prepared by immunizing an animal with a binding protein or CDR-containing region thereof. The immunized animal recognizes and reacts to the idiotypic determinants of the immunized antibody and produces anti-Id antibodies. Anti-Id antibodies can also be used as “immunogens” to induce an immune response in another animal, which produces so-called anti-anti-Id antibodies.

6). Use of the present antibodies Given their ability to bind to human RGM A, using conventional immunoassays such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or tissue immunohistochemistry (eg In order to detect human RGM A (in biological samples such as serum or plasma), the neutralizing antibody of the present application or a part thereof can be used. In the present application, the antibody or antibody portion of the present invention is contacted with a biological sample, and either the antibody (or antibody portion) or unbound antibody (or antibody portion) that binds to human RGM A is detected, and thereby in the biological sample. There is provided a method for detecting human RGM A in a biological sample comprising detecting human RGM A. The antibody is directly or indirectly labeled with a detectable substance to facilitate detection of bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; Examples of such fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; examples of luminescent materials include luminol; Examples of materials include ³ H, ¹⁴ C, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹²⁵ I, ¹³¹ I, ¹⁷⁷ Lu, ¹⁶⁶ Ho, ¹⁵³ Sm.

  The antibodies and antibody portions of the present application are preferably capable of neutralizing human RGM A activity both in vitro and in vivo. Thus, such antibodies and antibody portions of the present invention inhibit RGM A binding to its receptor neogenin, to BMP-2 and / or BMP-4, and thus to inhibit the resulting activity. Can be used.

  In another embodiment, the application provides a method for reducing RMG A activity in a subject, advantageously from a subject suffering from a disease or disorder in which the activity resulting from RGM A is detrimental. The present application relates to RMG A in subjects suffering from such diseases or disorders by blocking RGM A binding to neogenin and / or BMP-2 and / or BMP-4 through the use of the monoclonal antibodies of the present application. Methods for reducing activity are provided. For therapeutic purposes, the antibodies of the present application, particularly the humanized antibodies disclosed herein, can be administered to a human subject. Furthermore, the antibodies of the present application can be administered to non-human mammals that express RGM A to which the antibodies can bind for veterinary purposes or as animal models of human disease. With respect to the latter, such animal models can be useful for assessing the therapeutic efficacy of the antibodies of the invention (eg, testing of dosage and time course of administration). As used herein, the term “disease in which RGM A activity is detrimental” refers to the presence of RGM A in a subject suffering from the disease or the resulting activity in the pathophysiology of the disease. It is intended to include diseases and other diseases that have been shown or expected to be either factors involved or factors contributing to the worsening of the disease. Thus, a disease in which RGM A activity is detrimental is a disease in which a decrease in RGM A activity is expected to alleviate the symptoms and / or progression of the disease. Non-limiting examples of diseases that can be treated with the antibodies of the present invention include those diseases discussed in the following sections relating to pharmaceutical compositions of the antibodies of the present invention.

  It has been recognized that RGM A plays an important role in pathologies associated with a variety of diseases, including neurological diseases associated with neurodegeneration or inhibition of the nerve regeneration process (resulting in paralysis). This includes dementia, senile dementia, mild cognitive impairment, Alzheimer-related dementia, Huntington's chorea, tardive dyskinesia, hyperactivity, depression, Parkinson's disease, Steel-Richard syndrome, Down's syndrome, myasthenia gravis , Neurotrauma, vascular amyloidosis, cerebral hemorrhage I with amyloidosis, encephalitis, Friedreich ataxia, acute confusion disorder, glaucoma, Alzheimer's disease, amyotrophic lateral sclerosis, brachial plexus injury, brain injury (trauma Cerebral palsy, Guillain Valley, cerebral white matter atrophy, multiple sclerosis, post-polio syndrome, spina bifida, spinal cord injury, spinal muscular atrophy, spinal tumor, stroke, transverse myelitis included.

  Also, as already discussed, DVD immunoglobulins or bispecific antibodies between any one of the above mentioned partners may be useful. Such antibody preparations as described above can be found in Alzheimer's disease, Parkinson's disease, spinal cord injury, traumatic brain injury, multiple sclerosis, peripheral nerve injury, schizophrenia, depression, anxiety and any plastic and neurite outgrowth and above It may be useful for the treatment of neurotoxicity related diseases cited in.

  The antibodies of the present application can also be combined with peptides that allow movement across the membrane to include targeting intracellular target proteins. Such peptide sequences can include, but are not limited to, tat, antennapedia, poly-arginine, and some antimicrobial peptides. Such peptides may allow movement through the membrane, including the cell plasma membrane as well as the epithelium and the inner membrane (including the vascular brain barrier, intestinal mucosa, meninges, etc.).

  The antibodies or antibody portions of the present application can also be administered with one or more additional small molecule therapeutics useful for the treatment of diseases involving RGM A activity as discussed in the preceding paragraph. It is understood that the antibodies or antigen-binding portions thereof of the present application can be used alone or in combination with further agents, such as therapeutic agents, which further agents are selected by the skilled worker for their intended purpose. I want to be. For example, the additional agent can be a therapeutic agent recognized in the art as useful for treating a disease or condition that is treated by the antibody of the present invention. The additional agent can also be an agent that imparts beneficial properties to the therapeutic composition, such as an agent that affects the viscosity of the composition.

7). Pharmaceutical Composition The present invention also provides a pharmaceutical composition comprising the antibody of the present invention or an antigen-binding portion thereof and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising an antibody of the present invention is used, but not limited to, in the diagnosis, detection or monitoring of a disease, in the prevention, treatment, management or amelioration of a disease or one or more symptoms thereof and / or in research Is for. In a specific embodiment, the composition comprises one or more antibodies of the invention. In another embodiment, the pharmaceutical composition comprises one or more antibodies of the invention and one or more prophylactic or therapeutic agents other than the antibodies of the invention for treating a disease in which RGM A activity is detrimental; including. Preferably, it is a prophylactic or therapeutic agent that is known or has been used or is currently used in the prevention, treatment, management or amelioration of a disease or one or more symptoms thereof. According to these embodiments, the composition may further comprise a carrier, diluent or excipient.

  The antibodies and antibody portions of the invention can be incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, the pharmaceutical composition comprises an antibody or antibody portion of the invention and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, and the like. Examples include tonicity agents and absorption delaying agents. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers can further include minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffering agents that enhance the shelf life or effectiveness of the antibody or antibody portion.

In order to administer one or more antibodies of the invention or combinations of one or more antibodies of the invention and prophylactic or therapeutic agents useful for the prevention, management, treatment or amelioration of a disease or one or more symptoms thereof Delivery systems are known and can be used (eg, liposomes, microparticles, encapsulation in microcapsules, recombinant cells capable of expressing antibodies or antibody fragments, receptor-mediated endocytosis (eg, Wu and Wu) J. Biol. Chem. 262: 4429-4432 (1987)), nucleic acid constructs as part of retroviruses or other vectors, etc.). The method of administering the prophylactic or therapeutic agent of the present invention is not limited to the following, but parenteral administration (eg, intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural administration, intratumoral administration And mucosal administration (eg, intranasal and buccal routes). In addition, pulmonary administration may be used, for example, by use of an inhaler or nebulizer and formulation with an aerosolizing agent. For example, US Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540 and 4,880,078; and PCT publications WO92 / 19244, WO97 / 32572, WO97 / 44013, WO98 / 31346 and WO99 / 66903 (these Each of which is incorporated herein by reference in their entirety.) In certain embodiments, Alkermes ^{AIR (R)} pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass .) Using the antibody of the present invention, the combination therapy, or a composition of the invention is administered. In a specific embodiment, the prophylactic or therapeutic agent of the present invention is administered intramuscularly, intravenously, intratumorally, orally, intranasally, intrapulmonary or subcutaneously. Prophylactic or therapeutic agents may be administered by any conventional route, for example by infusion or bolus injection, by absorption through epithelial or dermal mucosal linings (eg oral mucosa, rectum and small intestinal mucosa, etc.) It can be administered with the active substance. Administration can be systemic or local.

In a specific embodiment, it may be desirable to administer the prophylactic or therapeutic agent of the present invention locally to the area in need of treatment; for example, but not limited to, local infusion Accordingly, by injection, or by implantation device (the implant, porous or non-porous material, including membranes and matrices, for example, silastic membranes, polymers, fibrous matrices (e.g., Tisseel ^(R)), or collagen matrices, etc. Can be achieved). In certain embodiments, an effective amount antagonist of one or more antibodies of the present invention is administered locally to a lesion in a subject to prevent, treat, manage and / or ameliorate the disease or its symptoms. In another embodiment, an effective amount of one or more antibodies of the invention is one or more therapeutic agents other than the antibodies of the invention to prevent, treat, manage and / or ameliorate the disease or one or more symptoms thereof. In combination with an effective amount (eg, one or more prophylactic or therapeutic agents) is administered locally to the lesion.

  In another embodiment, the prophylactic or therapeutic agent can be delivered in a controlled release or sustained release system. In certain embodiments, a pump may be used to effect controlled or sustained release (Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:20; Buchwald et al., 1980, Surgary 88: 507; Saudek et al., 1989, N. Engl. J. Med. 321: 574). In another embodiment, polymeric materials can be used to provide controlled or sustained release of the therapeutics of the invention (see, eg, Medical Applications of Controlled Release, Langer and Wise (ed.), CRC Pres., Robo, Fla. (1974); Chem., 23:61; see also Levy et al., 1985, Science 2. During et al., 1989, Ann. Neurol. 25: 351; Howard et al., 1989, J. Neurosurg. 71: 105); U.S. Pat. No. 5,679,377; 5,916,597; No. 5,912,015; No. 5,989,463; No. 5,128,326; see also PCT publication WO 99/15154; and PCT publication WO 99/20253). Examples of polymers used in sustained release formulations include, but are not limited to, poly (2-hydroxyethyl methacrylate), poly (methyl methacrylate), poly (acrylic acid), poly (ethylene-vinyl acetate) copolymers, poly (Methacrylic acid), polyglycolide (PLG), polyanhydride, poly (N-vinylpyrrolidone), poly (vinyl alcohol), polyacrylamide, poly (ethylene glycol), polylactide (PLA), poly (lactide-glycolide) copolymer (PLGA) and polyorthoesters are included. In a preferred embodiment, the polymer used in the sustained release formulation is inert, free of eluting impurities, stable on storage, sterilized, and biodegradable. In yet another embodiment, a controlled release or sustained release system can be placed in close proximity to the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose (eg, Goodson, Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

  Controlled release systems are discussed in overview by Langer (1990, Science 249: 1527-1533). Any technique known to those skilled in the art can be used to prepare a sustained release formulation comprising one or more therapeutic agents of the present invention. For example, U.S. Pat. No. 4,526,938, PCT publication WO 91/05548, PCT publication WO 96/20698, Ning et al., 1996, “Intratumor Radioimmunotherapy of a Human Colon Xenograft Usa Re-Gene O & G” 179-189, Song et al., 1995, “Antibody Mediated Lung Targeting of Long-Circulating Emulations”, PDA Journal of Pharmaceutical Science & Technol. 997, "Biodegradable polymeric Carriers for a bFGF Antibody for Cardiovascular Application", Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24: 853-854 and Lam et al., 1997, “Microencapsulation of Recombinant Humanized Monoclonal Antibody for Local Delivery”, Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24: 759-760, each of which is incorporated herein by reference in their entirety.

  In a specific embodiment, if the composition of the invention is a nucleic acid encoding a prophylactic or therapeutic agent, it is constructed as part of a suitable nucleic acid expression vector and administered so that it enters the cell. By, for example, using retroviral vectors (see US Pat. No. 4,980,286) or by direct injection or by using particle bombardment (eg, gene gun; Biolistic, Dupont) or lipid or cell surface receptors Alternatively, by coating it with a gene transfer agent or by linking it to a homeobox-like peptide known to enter the nucleus (eg, Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88). 1864-1868), the encoded preventive drug or Nucleic acid can be administered in vivo to promote expression of the therapeutic agent. Alternatively, the nucleic acid can be introduced into the cell and incorporated into the host cell DNA for expression by homologous recombination.

  A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include, but are not limited to, parenteral, such as intravenous, intradermal, subcutaneous, oral, nasal (eg inhalation), transdermal (eg topical), transmucosal and rectal administration. In a specific embodiment, the composition is formulated according to normal procedures as a pharmaceutical composition suitable for intravenous, subcutaneous, intramuscular, oral, nasal or topical administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If desired, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to relieve pain at the injection site.

  If the composition of the present invention is to be administered topically, it is formulated in an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion or other form well known to those skilled in the art. be able to. For example, Remington's Pharmaceutical Sciences and Induction to Pharmaceutical Dosage Forms, 19th Edition, Mack Pub. Co. Easton, Pa. (1995). For topical dosage forms that are not for spraying, semi-solid or solid form viscosities that are compatible with topical application and preferably have a higher dynamic viscosity than water or one or more excipients are usually used. . Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, coatings, salves, etc., where appropriate, they can be sterilized or It is mixed with auxiliary substances (for example preservatives, stabilizers, wetting agents, buffers or salts) to influence various properties, for example osmotic pressure. For other suitable topical dosage forms, preferably in combination with a solid or liquid inert carrier, the active ingredient in a mixture with a pressurized volatile (eg a gas propellant such as freon) or in an squeeze bottle An aerosol formulation for spraying is included. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms as needed. Examples of such additional components are well known in the art.

  Where the method of the invention involves intranasal administration of the composition, the composition can be formulated in aerosol form, spray, mist or in the form of drops. In particular, prophylactic or therapeutic agents for use according to the present invention conveniently use suitable propellants (eg, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). Can be delivered in the form of an aerosol spray from a pressurized pack or a nebulizer. In the case of a pressurized aerosol, the dosage unit can be determined by providing a value to deliver a metered amount. Capsules and cartridges (eg consisting of gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch. .

  Where the method of the invention includes oral administration, the composition can be formulated for oral use in the form of tablets, capsules, cachets, gelcaps, solutions, suspensions, and the like. Binders (eg pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (eg lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (eg magnesium stearate, talc or silica); disintegration Tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as an agent (eg, potato starch or sodium starch glycolate); or a humectant (eg, sodium lauryl sulfate). Tablets can be coated by methods well known in the art. Liquid formulations for oral administration can take the form of, but are not limited to, solutions, syrups or suspensions, or they can be used as a dry product for constitution with water or other suitable vehicle prior to use. Can be given. Suspensions (eg sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifiers (eg lecithin or acacia); non-aqueous vehicles (eg almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and storage Such liquid formulations may be prepared by conventional means, with pharmaceutically acceptable additives such as a material (eg, methyl or propyl-p-hydroxybenzoate or sorbic acid). The formulation may also contain buffer salts, flavoring agents, pigments and sweetening agents as desired. Formulations for oral administration may be suitably formulated for sustained release, controlled release or sustained release of prophylactic or therapeutic agents.

The methods of the invention can include pulmonary administration, eg, by use of an inhaler or nebulizer of a composition formulated with an aerosolizing agent. For example, US Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540 and 4,880,078; and PCT publications WO92 / 19244, WO97 / 32572, WO97 / 44013, WO98 / 31346 and WO99 / 66903 (these Each of which is incorporated herein by reference in their entirety.) In a specific embodiment, compositions of an antibody, combination therapy, and / or the invention of the present invention, Alkermes ^{AIR (R)} pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass .) Is administered with The

  The methods of the invention may include administration of a composition formulated for parenteral administration by injection (eg, by bolus injection or continuous infusion). Formulations for injection can be given in unit dosage forms (eg, in ampoules or multi-use containers) supplemented with preservatives. The composition may take the form of a suspension, solution or emulsion in oil or an aqueous vehicle, and also contains a formulation agent necessary for formulation such as a suspension, stabilizer and / or dispersant. obtain. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle (eg, sterile pyrogen-free water) prior to use. The methods of the present invention may further comprise administration of a composition formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the composition is formulated with a suitable polymeric or hydrophobic material (eg, as an acceptable emulsion in oil) or with an ion exchange resin, or as a poorly soluble derivative (eg, as a poorly soluble salt). Can be done.

  The methods of the present invention include administration of compositions formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and sodium, potassium, ammonium, calcium, iron hydroxide, isopropylamine , Triethylamine, 2-ethylaminoethanol, histidine, procaine and the like formed with cations.

  In general, the components of the composition are mixed individually or in unit dosage form, for example as a lyophilized powder or water-free concentrate in a sealed container such as an ampoule or sachet indicating the amount of active substance. Supplied. Where the mode of administration is infusion, the composition can be formulated with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the method of administration is by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

  In particular, the present invention also provides that one or more of the prophylactic or therapeutic agent or pharmaceutical composition of the present invention is enclosed in a sealed container such as an ampoule or sachet that displays the amount of the drug. In certain embodiments, one or more of the prophylactic or therapeutic agents or the pharmaceutical composition of the present invention is provided as a dry sterile lyophilized powder or water-free concentrate in a sealed container for administration to a subject. Can be reconstituted to an appropriate concentration (eg, with water or saline). Preferably, one or more of the prophylactic or therapeutic agents or the pharmaceutical composition of the present invention is at least 5 mg, more preferably at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, at least 75 mg or at least 100 mg. Unit doses are supplied as sterile lyophilized powders dried in sealed containers. The freeze-dried prophylactic or therapeutic agent or the pharmaceutical composition of the present invention should be stored in its original container between 2 ° C. and 8 ° C., and the prophylactic or therapeutic agent or the pharmaceutical composition of the present invention. Is administered within 1 week after reconstitution, preferably within 5 days, within 72 hours, within 48 hours, within 24 hours, within 12 hours, within 6 hours, within 5 hours, within 3 hours or within 1 hour Should. In an alternative embodiment, one or more of the prophylactic or therapeutic agents or the pharmaceutical composition of the invention is supplied in liquid form in a sealed container indicating the amount and concentration of the drug. Preferably, the liquid form of the composition administered is at least 0.25 mg / mL, more preferably at least 0.5 mg / mL, at least 1 mg / mL, at least 2.5 mg / mL, at least 5 mg / mL, at least 8 mg / mL. Supplied in a sealed container at mL, at least 10 mg / mL, at least 15 mg / kg, at least 25 mg / mL, at least 50 mg / mL, at least 75 mg / mL or at least 100 mg / mL. The liquid form should be stored between 2 ° C and 8 ° C in its original container.

The antibodies and antibody portions of the invention can be incorporated into pharmaceutical compositions suitable for parenteral administration. Preferably, the antibody or antibody portion is prepared as an injectable solution containing 0.1-250 mg / mL antibody. Injectable solutions can be comprised of either liquid or lyophilized formulations in flint glass or brownish vials, ampoules or prefilled syringes. The buffer can be L-histidine (1-50 mM, optimally 5-10 mM) at a pH of 5.0 to 7.0 (optimally pH 6.0). Other suitable buffers include, but are not limited to, sodium succinate, sodium citrate, sodium phosphate or potassium phosphate. Sodium chloride can be used to change the toxicity of the solution at a concentration of 0-300 mM (optimally 150 mM for liquid dosage forms). For lyophilized formulations, a cryoprotectant, essentially 0-10% sucrose (optimally 0.5-1.0%) may be included. Other suitable cryoprotectants include trehalose and lactose. In the case of lyophilized dosage forms, a filler, essentially 1-10% mannitol (optimally 2-4%) may be included. Stabilizers can basically use 1-50 mM L-methionine (optimally 5-10 mM) in both liquid and lyophilized dosage forms. Other suitable fillers include glycine and arginine and may be included as 0-0.05% polysorbate-80 (optimally 0.005-0.01%). Additional surfactants include, but are not limited to, polysorbate 20 and BRIJ surfactants. Pharmaceutical compositions comprising the antibodies and antibody portions of the invention prepared as injectable solutions for parenteral administration include those used to facilitate absorption or dispersion of therapeutic proteins (eg, antibodies), etc. Additional materials useful as adjuvants can be included. Particularly useful adjuvant, Hylenex ^(R) (recombinant human hyaluronidase) is hyaluronidase, such as. Addition of hyaluronidase to an injectable solution improves human bioavailability after parenteral administration, particularly subcutaneous administration. This also makes it possible to increase the volume of the injection site (ie greater than 1 mL) while reducing pain and discomfort and minimizing the occurrence of an injection site reaction (WO 04/078140 and published US patent applications). US 2006104968 (incorporated herein by reference).

  The compositions of the present invention can be in various forms. These include, for example, liquid, semi-solid and solid dosage forms such as solutions (eg, injection and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Commonly preferred compositions are in the form of injectable or infusion solutions, such as those similar to those used for human passive immunization with other antibodies. The preferred method of administration is parenteral (eg, intravenous, subcutaneous, intraperitoneal, intramuscular). In preferred embodiments, the antibody is administered by intravenous infusion or injection. In another preferred embodiment, the antibody is administered by intramuscular or subcutaneous injection.

  A therapeutic composition usually must be sterile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. A sterile injectable solution is made by formulating the active compound (ie, antibody or antibody portion) in the required amount, in an appropriate solvent, optionally with a combination of one or more of the ingredients listed above, followed by It can be prepared by filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile lyophilized powders for preparing sterile injectable solutions, the preferred method of preparation is to obtain a powder obtained by adding any additional desired ingredients to the active ingredient from these pre-sterilized solutions by vacuum drying and spray drying. It is. The proper fluidity of a solution can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion and by using a surfactant. By including agents that delay absorption in the composition, for example, monostearate salts and gelatin, the injectable composition can be made to absorb continuously.

  The antibodies and antibody portions of the invention can be administered by a variety of methods known in the art, but for many therapeutic applications, the preferred route / method of administration is subcutaneous injection, intravenous injection or infusion. It is. It will be appreciated by those skilled in the art that the route and / or method of administration will vary depending on the desired result. In certain embodiments, the active compounds can be prepared with carriers that will not release the compound rapidly, such as controlled release formulations, including implants, transdermal patches, and microcapsule delivery systems. Biodegradable and biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. For example, Sustained and Controlled Release Drug Delivery Systems, J. Org. R. Edited by Robinson, Marcel Dekker, Inc. , New York, 1978.

  In certain embodiments, an antibody or antibody portion of the invention can be orally administered, for example, with an inert diluent or an assimilable edible carrier. This compound (and other ingredients as needed) can also be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or mixed directly into the subject's diet. In the case of oral administration of a therapeutic agent, this compound can be blended with excipients and used in the form of orally ingested tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. In order to administer a compound of the present invention by other than parenteral administration, it may be necessary to coat the compound with a substance that prevents its inactivation or to administer the compound with them.

  Supplementary active substances can also be incorporated into the compositions. In certain embodiments, the antibody or antibody portion of the invention is co-formulated and / or co-administered with one or more additional therapeutic agents that are useful for treating a disease in which RGM A activity is detrimental. . For example, the anti-RGM A antibody or antibody portion thereof of the invention can be co-formulated with one or more additional antibodies that bind to other targets (eg, antibodies that bind to cytokines or bind to cell surface molecules) and / or Or they can be co-administered. Furthermore, one or more antibodies of the invention can be used in combination with two or more of the preceding therapeutic agents. Such combination therapies can advantageously use lower doses of the therapeutic agent administered, thus avoiding the potential for toxicity or complications associated with various monotherapy.

  In certain embodiments, the antibody against RGM A or a fragment thereof is linked to a half-life extending vehicle known in the art. Such vehicles include, but are not limited to, the Fc domain, polyethylene glycol, and dextran. Such vehicles are described, for example, in US patent application Ser. No. 09 / 428,082 and published PCT application WO 99/25044, which are hereby incorporated by reference for all purposes.

  In a specific embodiment, a nucleotide sequence encoding an antibody of the invention or another prophylactic or therapeutic agent of the invention to treat, prevent, manage or ameliorate a disease or one or more symptoms thereof by gene therapy. A nucleic acid sequence comprising is administered. Gene therapy refers to a therapy performed by administration to a subject of an expressed or expressible nucleic acid. In this embodiment of the invention, the nucleic acid produces their encoded antibody or a prophylactic or therapeutic agent of the invention that mediates a prophylactic or therapeutic effect.

  In accordance with the present invention, any of the methods for gene therapy available in the art can be used. For a general review of gene therapy methods, see Goldspiel et al., 1993, Clinical Pharmacy 12: 488-505; Wu and Wu, 1991, Biotherapy 3: 87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32: 573-596; Mulligan, Science 260: 926-932 (1993); and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62: 191-217; May 1993, TIBTECH 11 (5): 155-215. Methods commonly known in the art of recombinant DNA technology that can be used are Ausubel et al. (Eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Era Manual, Stockton Press, NY (1990). A detailed description of various methods of gene therapy is disclosed in US Patent Application Publication No. US20050042664 A1, which is incorporated herein by reference.

  RGM A plays an important role in the pathology associated with various diseases as defined herein above. RGM A and RGM protein are found in the peri-infarct region of human brain affected by stroke at the site of human injury suffering from traumatic brain injury (Schwab et al., Arch. Neurol. 62: 1561-8, 2005a). And in the core region (Schwab et al., Arch. Neurol. 62: 1561-8, 2005a), in the substantia nigra of patients suffering from Parkinson's disease (Bossers et al., Brain Pathology vol. 19: 91-107, 2008), It is described that it is up-regulated. Thus, RGM A antibody is a suitable agent for combination therapy of stroke, traumatic brain injury, Parkinson's disease, Alzheimer's disease and other neurodegenerative diseases of the human nervous system. In stroke patients, the latest treatment within the first 3 hours consists of the delivery of tissue plasminogen activator for clot lysis (Liang et al., Arch. Neurol. 65: 1429-33, 2008) Such treatments can in principle be combined with RGM A antibody delivery, resulting in different therapeutic approaches and expanding further therapeutic window. In Alzheimer's disease, drug combinations with RGM A antibodies are possible with approved cognitive enhancers, donepezil, and memantine, and such an approach can significantly suppress the progression of progressive neuropathology. Intranasal delivery of insulin has a positive effect on attention and memory (Hanson and Frey, BMC Neurosci. 9: S5, 2008) and is a possible route of administration for RGM A antibodies, thereby allowing the blood-brain barrier To detour. In Parkinson's disease (PD) patients, the latest treatments are mainly dopamine agonists such as levodopa, dopamine prodrugs (Khor and Hsu, Curr. Clin. Pharmacol. 2: 234-43, 2007), ropinirole, non-ergoline. System dopamine agonists (Jost et al., J. Neurol. 255 Suppl. 5: 60-63, 2008), monoamine oxidase B inhibitors rasagiline and selegrins (Elmer and Bertoni, Expert Opin. Pharmacother. 9: 2759-72, 2008). Based. Despite their beneficial effects in early and mild PD, none of these drugs can prevent progressive degeneration in the substantia nigra and related subcortical and cortical areas, thus regenerating stimuli Combination therapy with sex RGM A antibodies can delay the course of the disease.

  Any neuroprotective agent, which is an anticonvulsant such as an antioxidant, radical scavenger, phenytoin or anemia, erythropoietin, is suitable for combination therapy with a pro-regenerative RGM A antibody, which usually results in a very short neuroprotection Expands the scope of therapeutic treatment of drugs.

  The antibodies and antibody portions of the invention can be used to treat humans suffering from such diseases.

  The antibodies of the invention or antigen-binding portions thereof can be used alone or in combination with further agents, such as therapeutic agents, which are selected by the skilled worker for their intended purpose. Please understand that. For example, the additional agent can be a therapeutic agent recognized in the art as useful for treating a disease or condition being treated by an antibody of the invention. The additional agent can also be an agent that confers an advantageous attribute to the therapeutic composition, eg, an agent that provides the viscosity of the composition. It should be further understood that the combinations to be included within the present invention are useful combinations for those applications. The agents described below are for illustrative purposes and are not intended to be limiting. These combinations that are part of the invention can be an antibody of the invention and at least one additional agent selected from the list below. The combination can also include a plurality of additional agents, such as two or three additional agents, if the combination is such that the composition formed can perform its desired function.

  Non-limiting examples of therapeutic agents for multiple sclerosis to which antibodies or antibody portions of the invention can be combined include the following: corticosteroids; prednisolone; methylprednisolone; azathioprine; cyclophosphamide; cyclosporine; -Aminopyridine; tizanidine; interferon-β1a (AVONEX; Biogen); interferon-β1b (BETASERON; Chiron / Berlex); interferon α-n3) (Interferon Sciences / Fujimomoto), interferon-α (AlfaJs / AlmanA / β) -IF (Serono / Inhale Therapeutics), pegylated interferon α2b (Enzon / Sc ering-Plough), copolymer 1 (Cop-1; COPAXONE; Teva Pharmaceutical Industries, Inc.); hyperbaric oxygen; intravenous immunoglobulin; cladribine; other human cytokines or growth factors and their receptors, eg, TNF, LT , IL-1, IL-2, IL-6, IL-7, IL-8, IL-23, IL-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF and PDGF Or an antagonist is included. The antibodies of the invention or antigen-binding portions thereof are directed against cell surface molecules such as CD2, CD3, CD4, CD8, CD19, CD20, CD25, CD28, CD30, CD40, CD45, CD69, CD80, CD86, CD90 or their ligands. Can be combined with antibodies. The antibody of the present invention or an antigen-binding portion thereof also contains methotrexate, cyclosporine, FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs such as corticosteroids such as ibuprofen and prednisolone, phosphodiesterase inhibitors, adenosine agonists, antithrombotic agents Agents that block signal transduction by inflammatory cytokines such as complement inhibitors, adrenergic agonists, TNFα or IL-1 (eg, IRAK, NIK, IKK, p38 or MAP kinase inhibitors), IL- 1β converting enzyme inhibitor, TACE inhibitor, T cell signaling inhibitor such as kinase inhibitor, metalloproteinase inhibitor, sulfasalazine, azathioprine, 6-mercaptopurine, angiotensin converting enzyme Inhibitors, soluble cytokine receptors and derivatives thereof (eg soluble p55 or p75 TNF receptor, sIL-1RI, sIL-1RII, sIL-6R) and anti-inflammatory cytokines (eg IL-4, IL-10, IL-13 and TGFβ) can also be combined.

  Preferred examples of therapeutic agents for multiple sclerosis to which an antibody or antigen-binding portion thereof can be combined include interferon-β, such as IFNβ1a and IFNβ1b; copaxone, corticosteroids, caspase inhibitors, such as inhibitors of caspase-1. , IL-1 inhibitors, TNF inhibitors and antibodies to CD40 ligand and CD80.

  The antibodies of the present invention or antigen-binding portions thereof are also alemtuzumab, dronabinol, unimed, daclizumab, mitoxantrone, xaliproden hydrochloride, fampridine, glatiramer acetate, natalizumab, cinnavidol, a-immunokine NNS03, ABR-215062, Anergix . MS, chemokine receptor antagonist, BBR-2778, caragualine, CPI-1189, LEM (liposome-encapsulated mitoxantrone), THC. CBD (cannabinoid agonist) MBP-8298, mesoplan (PDE4 inhibitor), MNA-715, anti-IL-6 receptor antibody, neurobax, pirfenidone allotrap 1258 (RDP-1258), sTNF-R1, taran panel, teriflunomide , TGF-β2, tiprimotide, VLA-4 antagonists (eg, TR-14035, VLA4 Ultrahaler, Antegran-ELAN / Biogen), interferon gamma antagonists, IL-4 agonists and other agents.

  A pharmaceutical composition of the invention may comprise a “therapeutically effective amount” or “prophylactically effective amount” of an antibody or antibody portion of the invention. “Therapeutically effective amount” refers to an amount effective to achieve the desired therapeutic result for the required period of time at the required dosage. The therapeutically effective amount of an antibody or antibody portion can be determined by one skilled in the art, such as the disease stage, age, sex and weight of an individual, the ability of the antibody or antibody portion to elicit a desired response in that individual, etc. Can vary according to other factors. A therapeutically effective amount is also such that the therapeutically beneficial effect exceeds any toxic or deleterious effects of the antibody or antibody portion. “Prophylactically effective amount” refers to an amount effective to achieve the desired prophylactic result for the required period of time at the required dosage. Usually, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

  Dosage regimens may be adjusted to provide the optimum desired response (eg, a therapeutic or prophylactic response). For example, a single bolus can be administered, several divided doses can be administered over a period of time, or the dose can be evenly reduced or increased as indicated by the requirements of the treatment situation . It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form, as used herein, refers to a physically separated unit suitable as a single dose for the mammalian subject to be treated; each unit is a required pharmaceutical Contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the carrier. The definition for the dosage unit form of the present invention is the technical field combining (a) the unique characteristics of the active compound and the specific therapeutic or prophylactic effect to be achieved and (b) the treatment of sensitivity in an individual. And are directly dependent on these.

  A typical non-limiting range for a therapeutically or prophylactically effective amount of an antibody or antibody portion of the invention is 0.1-20 mg / kg, more preferably 1-10 mg / kg. Note that dosage values can vary depending on the type and severity of the condition to be alleviated. For any particular subject, the specific dosage regimen should be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the composition. It should be further understood that the dosage ranges set forth in are exemplary only and are not intended to limit the scope or practice of the claimed composition.

  It will be apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the invention described herein will be apparent and depart from the scope of the invention and the embodiments disclosed herein. Without using the appropriate equivalent. Having described the invention in detail, the invention will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to limit the invention. .

Method The following method details the experimental procedure used in the experimental section.

(I) Direct binding ELISA plates were coated with hRGM A (R & D) at a concentration of 2 μg / mL in carbonate buffer. Next, the well was blocked with a 2% blocking solution (Bio-Rad) for 1 hour at room temperature. From the top to the bottom of the plate, the biotinylated antibody was serially diluted at a dilution factor of 1: 5 in 0.1% BSA / PBS and incubated at room temperature for 1 hour. The detection reagent was a 1: 10,000 dilution of streptavidin-HRP in 0.1% BSA / PBS. Detection was with TMB reagent, stopped with 2N H ₂ SO ₄ and OD read at 450 nM.

  (Ii) FACS analysis. Stable gene transfectants of HEK293 cells overexpressing hRGM A or BAF3 cells overexpressing rat RGM A in 0.1% BSA / PBS buffer at 4 ° C. for> 15 min, unlabeled 5F9 or 8D1 MAB Stained. Detection was with mouse anti-rat IgG PE antibody.

(Iii) Solid phase ELISA assay to evaluate MAB 5F9 in hRGM A-neogenin binding assay At a concentration of 2.5 μg / mL of the extracellular domain of His-tagged human neogenin protein (stock solution concentration: 30 μg / mL) ELISA plates (Immuno Plate Cert. Maxi Sorb. F96 NUNC, 439454) were coated for 1 hour at 37 ° C. After incubation, unbound neogenin was removed in three separate washing steps with PBS containing 0.02% Tween20. The neogenin-coated plate was blocked by adding 200 μL / well of 3% bovine serum albumin (BSA), PBS, Tween 20 (0.02%) blocking solution. After incubation at 37 ° C. for 1 hour, the blocking solution was removed, and an RGM A fragment or full-length protein conjugated with a human fc tag was added with or without antibodies. In some experiments, antibodies were preincubated with fc-conjugated hRGM A protein for 1 hour at room temperature. Neogenin-coated plates with or without antibodies were incubated with hRGM A for 1 hour at 37 ° C. After three washing steps with PBS-Tween 20 (0.02%), diluted 1: 200 in PBS containing biotin-labeled anti-human fc antibody (1 mg / mL, 0.6% BSA, 0.02% Tween 20) ), Jackson ImmunoResearch catalog number: 709-065-149, and plates were incubated at 37 ° C. for 1 hour. Unbound antibody was removed by three washing steps using PBS-Tween 20 (0.02%). To visualize the binding of biotin-labeled anti-fc antibody, consists of streptavidin-peroxidase (Roche, cat. # 110899153001) diluted 1: 5000 in PBS containing 0.6% BSA, 0.02% Tween20 The complex was added followed by 1 hour incubation at 37 ° C. Prior to the addition of peroxidase substrate (Immuno Pure TMB, Pierce # 34021), unbound peroxidase complex was removed in three subsequent washing steps (PBS-Tween 20 (0.02%)). The substrate reaction was stopped with 2.5 MH ₂ SO ₄ 1-30 minutes after addition of substrate to the wells. The plate was analyzed (OD measurement) at a wavelength of 450 nm using an Anthos photometer.

(Iv) Solid phase ELISA assay to evaluate MAB 5F9 in hRGM A-BMP-4 binding assay. Recombinant human BMP-4 protein (R & D Systems, # 314-BP, Lot # BEM316061) at a concentration of 2.5 μg / mL. An ELISA plate (Immuno Plate Cert. Maxi Sorb. F96 NUNC, 439454) was coated with the contained solution for 1 hour at 37 ° C. After incubation, unbound BMP-4 was removed in three separate washing steps with PBS containing 0.02% Tween20. BMP-4 coated plates were blocked by adding 200 μL / well of 3% bovine serum albumin (BSA), PBS, Tween 20 (0.02%) blocking solution. After incubation at 37 ° C. for 1 hour, the blocking solution was removed, and an RGM A fragment or full-length protein bound to a human fc tag was added with or without an antibody. In some experiments, antibodies were preincubated with fc-conjugated hRGM A protein for 1 hour at room temperature. BMP-4 coated plates with hRGM A with or without antibodies were incubated for 1 hour at 37 ° C. After three washing steps with PBS-Tween 20 (0.02%), diluted 1: 200 in PBS containing biotin-labeled anti-human fc antibody (1 mg / mL, 0.6% BSA, 0.02% Tween 20) ), Jackson ImmunoResearch catalog number: 709-065-149, and plates were incubated at 37 ° C. for 1 hour. Unbound antibody was removed by three washing steps with PBS-Tween 20 (0.02%). To visualize the binding of biotin-labeled anti-fc antibody, consists of streptavidin-peroxidase (Roche, cat. # 110899153001) diluted 1: 5000 in PBS containing 0.6% BSA, 0.02% Tween20 The complex was added followed by incubation at 37 ° C. for 1 hour. Prior to the addition of peroxidase substrate (Immuno Pure TMB, Pierce # 34021), unbound peroxidase complex was removed in three successive washing steps (PBS-Tween 20 (0.02%)). The substrate reaction was stopped with 2.5 MH ₂ SO ₄ 1-30 minutes after addition of substrate to the wells. The plate was analyzed (OD measurement) at a wavelength of 450 nm using an Anthos photometer.

(V) Solid phase ELISA assay to evaluate MAB 5F9 in hRGM A-BMP-2 binding assay. Recombinant human BMP-2 protein (R & D Systems, # 355-BM, Lot # MSA04) at a concentration of 2.5 μg / mL. Using the contained solution, an ELISA plate (Immuno Plate Cert. Maxi Sorb. F96 NUNC, 439454) was coated for 1 hour at 37 ° C. After incubation, unbound BMP-2 was removed in three separate washing steps with PBS containing 0.02% Tween20. Blocking of BMP-2 coated plates was performed by adding 200 μL / well of 3% bovine serum albumin (BSA), PBS, Tween 20 (0.02%) blocking solution. After incubation at 37 ° C. for 1 hour, the blocking solution was removed and RGM A fragment or full-length protein conjugated with human fc tag, with or without antibody, was added. In some experiments, antibodies were preincubated with fc-conjugated hRGM A protein for 1 hour at room temperature. BMP-2 coated plates with or without antibodies were incubated with hRGM A for 1 hour at 37 ° C. After three washing steps with PBS-Tween 20 (0.02%), diluted 1: 200 in PBS containing biotin-labeled anti-human fc antibody (1 mg / mL, 0.6% BSA, 0.02% Tween 20) ), Jackson ImmunoResearch catalog number: 709-065-149, and plates were incubated at 37 ° C. for 1 hour. Unbound antibody was removed by three washing steps with PBS-Tween 20 (0.02%). To visualize the binding of biotin-labeled anti-fc antibody, consists of streptavidin-peroxidase (Roche, cat. # 110899153001) diluted 1: 5000 in PBS containing 0.6% BSA, 0.02% Tween20 The complex was added followed by incubation at 37 ° C. for 1 hour. Prior to the addition of peroxidase substrate (Immuno Pure TMB, Pierce # 34021), unbound peroxidase complex was removed in three successive washing steps (PBS-Tween 20 (0.02%)). The substrate reaction was stopped with 2.5 MH ₂ SO ₄ 1-30 minutes after addition of substrate to the wells. The plate was analyzed (OD measurement) at a wavelength of 450 nm using an Anthos photometer.

(Vi) Ntera-2 cell culture Human Ntera-2 cells were obtained from German Collection of Microorganisms and Cell Culture (DMSZ, Braunschweig). A frozen stock of undifferentiated Ntera-2 cells was freeze-thawed in DMEM medium containing 10% fetal bovine serum (FBS; JRH Bioscience, Kansas, USA) and 5% horse serum (HS; Sigma, Germany). Cells were grown in culture flasks (Greiner, Germany) until the cells reached 80% confluency.

For neural differentiation, Ntera − at a density of 2.5 × 10 ⁶ cells / 175 cm ² in differentiation medium (DMEM medium containing 10% FBS, 5% HS, 1% penicillin-streptomycin, 10 μm retinoic acid). Two cells were seeded. Cells were differentiated for 3 weeks and the medium was changed twice a week.

  After differentiation, the cells were detached with trypsin-EDTA and divided at a ratio of 1: 6. After 48 hours, neurons were separated from the underlying cells by tapping. The detached cells were transferred to a new shake culture flask (Corning, USA) for aggregation in fresh medium. Differentiated Ntera-2 cells were agglutinated in Neurobasal medium (Gibco) supplemented with B27 (Gibco), glutamine (Gibco) and penicillin-streptomycin for 24 hours at 37 ° C. under smooth horizontal shaking conditions. Ntera-2 aggregates were seeded at a density of approximately 20-30 aggregates per coverslip in a 24-well plate. Coverslips pre-coated with polylysine were coated with laminin (20 μg / mL, Sigma) and recombinant fc-linked human RGM A fragment # 786 (amino acids 47-168) at a concentration of 10 μg / mL. After seeding, the cultures were treated with 5F9 MAB (added to medium at 3 different concentrations (0.1 μg / mL; 1 μg / mL; 10 μg / mL)) and further incubated at 37 ° C. for 24 hours in Neurobasal medium. . The aggregates were then fixed in 4% paraformaldehyde (2 hours, room temperature) and permeabilized (20 minutes, room temperature) by addition of 0.1% Triton X-100 in PBS. For fluorescent staining, cultures were blocked with PBS containing 1% BSA for 1 hour at room temperature. After blocking treatment, Ntera cells were incubated for 2 hours at room temperature with a mouse monoclonal antibody against β-tubulin isotype 3 (clone SDL3D10, Sigma # T8660). Unbound antibody was removed by three separate washing steps (5-15 min each), and a Cy-3 conjugated donkey anti-antibody diluted 1: 350 in PBS / 0.5% BSA and 0.5 μg / mL bisbenzimide. Ntera cells were incubated with mouse antibody (Jackson ImmunoResearch Lot 62597). After 1 hour incubation, the culture was washed 3 times to remove unbound secondary antibody. Coverslips were embedded in Fluoromount G (Southern Biotech, Eching) for fluorescence microscopy.

  Images of Ntera-2 aggregates were collected using a Zeiss Axiovert 200 fluorescence microscope and culture elongation was analyzed automatically using an in-house image acquisition and analysis system. Automatic analysis of elongation was performed with Image Pro Plus 4.5 and statistical analysis of data was performed with Graph Pad Prism 4. Extension was normalized to a control culture grown in the absence of human RGM A fragment # 786.

(Vii) SH-SY5Y culture SH-SY5Y cells (ATCC, CRL-2266) are human neuroblastoma cells derived from metastatic brain tumors. # 31765-02 from 50% Earl Balanced Salt Solution (Invitrogen Life Technologies, Cat. # 24010-043) and 50% F12 (Ham) Nutrient Mix + GlutaMAX-1 (Invitrogen Life Technologies cells from Cat. # 31765-7). Allowed to grow. Heat-inactivated 10% fetal calf serum (FCS, JRH Biosciences, Kansas Cat. # 12107-1000M), 1% NEAA (MEM non-essential amino acid solution (Sigma-Aldrich Cat. # M1745)) and 1% penicillin (10.000 U) / ML) / streptomycin (10.000 μg / mL) (Invitrogen Life Technologies, Cat. # 15140-122) was added to this medium, 10 μm over several days to stimulate neuronal differentiation and neurite outgrowth. SH-SY5Y cells were cultured in a medium supplemented with retinoic acid (RA, Sigma-Aldrich Cat. # R2625-050MG). Differentiated SH-SY5Y cells were grown in tissue culture flasks, removed by careful trypsinization, and placed on glass coverslips coated with a stripe pattern of RGM A protein or fragments thereof and collagen I.

(Viii) Preparation of striped glass cover slips Modification of strip assay on glass cover slips in a slightly different manner than previously described (Knoell et al., Nature Protocols 2: 1216-1224, 2007). This is summarized below.

  A sterile silicon substrate for the production of stripes of purified protein was pressed onto the surface of the Petri dish with the rough side of the substrate facing up. A clean coverslip washed with ethanol is placed on this substrate and the corners of the substrate are marked with an ink ballpoint pen on the back of the coverslip. The base on which the cover slip was placed was carefully turned over so that the cover slip would face the bottom of the Petri dish. In order to visualize the RGM A stripe, FTC-binding full length inhibitory RGM A or Fc-fragment or recombinant human RGM A (R & D Systems Cat. Sigma-Aldrich Cat. # F-4018). Carefully inject 50 μL of RGM A-FITC antibody solution through the inlet using a Hamilton syringe. Excess liquid spilled from the base through the outlet, which is removed with a Kleenex cloth. After incubating the matrix-coverslip at 37 ° C. for 2 hours, the first coating solution (containing RGM A) was washed away with 100 μL of PBS. In the next step, cover slips with RGM A stripes are transferred to a 24-well plate and 500 μL collagen I (rat tail collagen I, Becton Dickinson Biosciences Cat. ) And incubated at 37 ° C. for 2 hours. Finally, an alternating stripe pattern of RGM A and collagen I was produced on the coverslip. After incubation, unbound collagen I was washed away in three separate washing steps with PBS and differentiated SH-SY5Y cells were placed on coverslips. Incubation of SH-SY5Y cells on patterned substrate in the presence or absence of monoclonal antibodies against human RGM A was continued at 37 ° C. for 20-24 hours.

  For immunofluorescence analysis, cells were fixed in 4% paraformaldehyde for 2 hours at room temperature or overnight at 4 ° C. and incubated with PBS containing 0.1% Triton X-100 for 10-20 minutes at room temperature. The permeation treatment was performed. After blocking with 3% BSA for 60 minutes, cells were incubated with primary antibody (monoclonal anti-β-tubulin isotype 3 clone SDL 3D10, Sigma-Aldrich Cat. # T8660) for 2 hours at room temperature, and several times After the washing step, the cells were incubated for 1 hour with a secondary antibody (Cy-3 donkey anti-mouse Jackson Immuno Research Lot: 62597) diluted in PBS containing 0.1% BSA. Nuclei were counterstained with bisbenzimide H33258 (Riedel-De-Haen, Cat. # A-0207). The cells were finally embedded in Fluoromount G (Southern Biotechnology Associates Inc .: Cat. # 010001). Cells were analyzed using an Axioplan 2 fluorescence microscope (Zeiss).

(Ix) Construction and expression of recombinant anti-RGM A antibody By homologous recombination in bacteria, the rat anti-human RGM A monoclonal antibodies 5F9 and 8D1 were transformed into a pHybE expression vector containing a human IgG1 constant region containing two hinge region amino acid mutations. The DNA encoding the heavy chain variable region cDNA fragment of was cloned. These mutations are leucine to alanine changes at positions 234 and 235 (EU numbering, Lund et al., 1991, J. Immunol., 147: 2657). The light chain variable regions of 5F9 and 8D1 monoclonal antibodies were cloned into a pHybE vector containing a human kappa constant region. Exemplary pHyb-E vectors include pHybE-hCk and pHybE-hCg1, z, non-a (see US Patent Application No. 61 / 021,282). Full-length antibodies were transiently expressed in 293E cells by simultaneous gene transfer of chimeric heavy and light chain cDNAs conjugated to pHybE expression plasmid. The cell supernatant containing the recombinant antibody was purified by protein A sepharose chromatography, and the bound antibody was eluted by the addition of an acidic buffer. The antibody was neutralized and dialyzed against PBS. The purified anti-human RGMA monoclonal antibodies were then tested for their ability to bind to RGM A by ELISA as described in Example 1 and competitive ELISA as described in Example 7.

Example 1
Preparation of anti-human RGM A monoclonal antibody Anti-human RGM A rat monoclonal antibody was obtained as follows.

Example 1A
Immunization of rats with human RGM A antigen On the first day, 25 μg of recombinant purified human RGM A (R & D Systems Cat # 2459-RM lot MRH02511A) mixed with complete Freund's adjuvant (Sigma) Harlan Sprague Dawley rats were injected subcutaneously. On days 21, 42 and 63, 25 μg of recombinant purified human RGM A mixed with incomplete Freund's adjuvant (Sigma) was injected subcutaneously into the same 4 Harlan Sprague Dawley rats. On days 144 or 165, rats were injected intravenously with 10 μg recombinant purified human RGM A.

(Example 1B)
Hybridoma Production To produce hybridomas, Kohler, G. et al. And in accordance with established methods described in Milstein 1975, Nature, 256: 495. Spleen cells obtained from the immunized rats described in A were fused with SP2 / O-cells in a 2: 1 ratio.

The fusion product was placed in a selective medium containing azaserine and hypoxanthine in 96-well plates at a density of 1.5 × 10 ⁵ spleen cells / well. Seven to ten days after the fusion, hybridoma colonies visible with the naked eye were observed. Supernatants from each well containing hybridoma colonies were tested by direct ELISA (see Example 2) for the presence of antibodies against human RGM A. ELISA positive cell lines were tested in FACS against stably transfected HEK293 cells expressing human and / or rat RGM A. Subsequently, these rat hybridoma cell lines were tested in a direct ELISA for cross-reactivity with rat RGM A and ELISA binding to the HuRGM A47-168 fusion protein.

(Example 2)
Direct ELISA binding of mAB5F9 and 8D1 As shown in FIG. 1A, MAB5F9 and 8D1 bind to hRGM A with similar titers as described in section (i) above. MAB 5F9 was also shown to bind to rat RGM A by ELISA, while 8D1 cannot bind to rat RGM A (data not shown). FIG. 1B shows that MAB5F9 and 8D1 bind to HEK293 cells that overexpress hRGM A in FACS. FIG. 1C shows that 5F9 (not 8D1) can bind to BAF3 cells overexpressing rat RGM A in FACS. FACS was performed as described in section (ii).

  A solid phase ELISA assay was used to assess MAB 5F9 binding in a competitive hRGM A-neogenin binding assay. ELISA plates were prepared and used as described in section (iii) of this application. HRGM A was added at a concentration of 0.5 μg / mL with 5F9 antibody for 1 hour at 37 ° C. Next concentrations: 1.25 μg / mL; 0.63 μg / mL; 0.32 μg / mL; 0.16 μg / mL; 0.08 μg / mL; 0.04 μg / mL; 0.02 μg / mL; 0.01 μg / mL MAB 5F9 was used in mL. The binding of hRGM A was visualized using a biotin-labeled anti-fc antibody and a streptavidin-peroxidase complex. Plates were analyzed using an Anthos photometer at a wavelength of 450 nm (OD measurement). As shown in FIG. 2, the three highest antibody concentrations inhibited full-length human RGM A binding to neogenin in a dose-dependent manner.

  A solid phase ELISA assay was also used to evaluate MAB 5F9 in a competitive hRGM A-BMP-4 binding assay. ELISA plates were prepared and used as described in section (iv) of this application. HRGM A was added at a concentration of 0.5 μg / mL with 5F9 antibody at 37 ° C. for 1 hour. Next concentrations: 1.25 μg / mL; 0.63 μg / mL; 0.32 μg / mL; 0.16 μg / mL; 0.08 μg / mL; 0.04 μg / mL; 0.02 μg / mL; 0.01 μg / mL MAB 5F9 was used in mL. The binding of hRGM A was visualized using a biotin-labeled anti-fc antibody and a streptavidin-peroxidase complex. Plates were analyzed using an Anthos photometer at a wavelength of 450 nm (OD measurement). As shown in FIG. 3, the highest four concentrations of antibody inhibited the binding of full-length human RGM A to BMP-4 in a dose-dependent manner.

  A solid phase ELISA assay was also used to assess MAB 5F9 binding inhibition of fragment 0 (47-168) hRGM A to BMP-4. ELISA plates were coated for 1 hour at 37 ° C. with a concentration of 2.5 μg / mL of human recombinant BMP-4 protein. HRGM A light chain (fragment 0, 47-168) was added at a concentration of 0.5 μg / mL at 37 ° C. for 1 hour with 5F9 antibody. Next concentrations: 1.25 μg / mL; 0.63 μg / mL; 0.32 μg / mL; 0.16 μg / mL; 0.08 μg / mL; 0.04 μg / mL; 0.02 μg / mL; 0.01 μg / mL MAB 5F9 was used in mL. The binding of hRGM A was visualized using a biotin-labeled anti-fc antibody and a streptavidin-peroxidase complex. Plates were analyzed using an Anthos photometer at a wavelength of 450 nm (OD measurement). FIG. 4 shows that antibody concentrations of 1.25 μg / mL, 0.63 μg / mL and 0.32 μg / mL inhibit human RGM A light chain binding to BMP-4 in a dose-dependent manner. A solid phase ELISA assay was also used to assess MAB 5F9 binding in the competitive hRGM A-BMP-2 binding assay. ELISA plates were prepared and used as described in section (v) of this application. Full length hRGM A was added at a concentration of 0.5 μg / mL together with 5F9 antibody at 37 ° C. for 1 hour. MAB 5F9 was used at the following concentrations: 5 μg / mL; 2.5 μg / mL; 1.25 μg / mL; 0.63 μg / mL; 0.32 μg / mL; The binding of hRGM A was visualized using a biotin-labeled anti-fc antibody and a streptavidin-peroxidase complex. Plates were analyzed using an Anthos photometer at a wavelength of 450 nm (OD measurement). FIG. 5 shows that antibody concentrations of 5 μg / mL, 2.5 μg / mL, 1.25 μg / mL, 0.63 μg / mL inhibit the binding of full-length human RGM A to BMP-2.

  A solid-phase ELISA assay was also used to evaluate MAB5F9 and 8D1 in the hRGM A-neogenin, hRGM A-BMP-2 and hRGM A-BMP-4 binding assays (FIG. 9). As described, ELISA plates were coated with an extracellular domain of His-tagged human neogenin protein at a concentration of 2.5 μg / mL or 2.5 μg / mL Bmp-2 or BMP-4 at 37 ° C. for 1 hour. Full length fc-conjugated hRGM A was added for 1 hour at 37 ° C. at a concentration of 0.5 μg / mL with the antibody. Use MAB 5F9 and 8D1 at the following concentrations: 5 μg / mL; 2.5 μg / mL; 1.25 μg / mL; 0.63 μg / mL; 0.32 μg / mL; 0.16 μg / mL; did. The binding of hRGM A was visualized using a biotin-labeled anti-fc antibody and a streptavidin-peroxidase complex. Plates were analyzed using an Anthos photometer at a wavelength of 450 nm (OD measurement). As shown in FIG. 9, rat monoclonal antibody 8D1 inhibits or reduces binding of human RGM A to BMP-2 and BMP-4, but cannot inhibit its binding to neogenin.

(Example 3)
MAb 5F9 activity in neurite outgrowth assays using aggregates of differentiated human NTera neurons Nter cells were obtained and cultured as described in method section (vi) of the present application. mAB 5F9 neutralized the neurite outgrowth inhibitory activity of the strong fc-linked light chain (amino acids 47-168) of the human RGM A protein in the neurite outgrowth assay by aggregation of differentiated human NTera neurons. As shown in FIG. 6, neuronal NTera aggregates in the absence of inhibitory RGM A protein or fragment and in the presence of the elongation-stimulating substrate laminin show a broad and dense network of extending neurites (A ). Also, as shown in FIG. 6, the presence of hRGM A light chain dramatically reduces the number, density and length of NTera neurites, demonstrating potent inhibitory activity of hRGM A fragments. . The few neurites that remain aggregated are short and tightly bundled (B). The CE part of FIG. 6 shows that the neurite outgrowth inhibitory activity of the hRGM A light chain fragment was neutralized or suppressed in a dose-dependent manner by increasing the concentration of MAB 5F9 added to the culture. With increasing concentrations of MAB, despite the presence of RGM A inhibitors (C: 0.1 μg / mL MAB 5F9; D: 1 μg / mL MAB 5F9; E: 10 μg / mL MAB 5F9), Ntera neuronal aggregates Elongation is fully maintained.

  To test a potent fc-binding light chain inhibitory fragment of human RGM A protein (amino acids 47-168), quantitative analysis of MAB 5F9 neutralizing activity in a neurite growth assay with human NTera aggregates was performed. Culture elongation was automatically analyzed by staining aggregates with bisbenzimide, followed by photography. This staining marked only aggregates, not elongating neurites. However, extending neurites were stained with an antibody against β3-tubulin and a fluorescently labeled secondary antibody. Neurite outgrowth was automatically measured by calculating the neurite outgrowth index (the index was calculated by subtracting the area of the cell body from the β3 tubulin stained area of the aggregate and its processes). Next, Lingor et al. Neurochem. 103: 181-189, 2007, this factor was divided by the cell body area. FIG. 7 shows that MAB 5F9 is dose dependent (0.1-10.0 μg) of fc-binding, potent hRGM A inhibitor fragment (fragment 0, 47-168; 10 μg) in a neurite outgrowth assay with human Ntera aggregates. It shows that the elongation inhibitory activity of

Example 4
mAb 5F9 activity in hRGM A / collagen I stripes SH-SY5Y cells were cultured and used as described in section (vii) of this application. Striped glass coverslips with RGM A and collagen I were prepared as described in section (viii) of this application. Carpets with alternating stripes of hRGM A / collagen I and collagen I were made for this experiment according to the protocol described in the literature (Knoell et al., Nature Protocols 2: 1216-1224, 2007). In the absence of 5F9 MAB (A), neuronal SH-SY5Y cells show clear selectivity for collagen I stripes, with 90% of these cells selecting collagen I stripes over hRGM A. As the concentration of MAB 5F9 increases, neuronal SH-SY5Y cells select the hRGM A stripe over the collagen I stripe (BE). At the highest MAB concentration used (E)), SH-SY5Y neurons show strong selectivity for hRGM A stripes compared to collagen I stripes (see FIG. 8). This can be interpreted as a unique feature of 5F9MAB, as 5F9MAB transformed the inhibitory nature of RGM A in its triggering activity. In the presence of increasing concentrations of 5F9, neurons tend to migrate to the RGM A substrate where they tend to grow and do not grow on permissive substrates such as collagen I. Such unique properties have not been previously described for monoclonal antibodies.

(Example 5)
Construction of CDR-grafted antibodies By applying standard methods well known in the art, the CDR sequences of the VH and VL chains of monoclonal antibody 5F9 (see Table 5 above) are converted to various human heavy and light chain acceptor sequences. Transplanted. Based on the sequence VH and VL alignment with the VH and VL sequences of the monoclonal antibody 5F9 of the present invention, the following known human sequences are selected:
a) VH3-48, VH3-33 and VH3-23 and the linking sequences hJH3, hJH4 and hJH6 to construct heavy chain acceptor sequences (according to Table 3 above);
b) A17 and A18 and hJK2 to construct the light chain acceptor sequence (according to Table 4 above).

  The following CDR grafted, humanized, modified VH and VL sequences were prepared by transplanting the corresponding VH and VL CDRs of 5F9 into the acceptor sequence (see also Table 6 above): VH5F9.1-GL, VH5F9 .2-GL, VH5F9.3-GL, VH5F9.4-GL, VH5F9.5-GL, VH5F9.6-GL, VH5F9.7-GL and VH5F9.8-GL; VL 5F9.1-GL, VL 5F9 2-GL and VL 5F9.3-GL.

(Example 6)
Construction of framework backmutations in CDR grafted antibodies To generate humanized antibody framework backmutations by de novo synthesis of variable domains and / or mutagenic primers and PCR and methods well known in the art Is used to introduce mutations into CDR grafted antibody sequences as prepared according to Example 5. Various combinations of back mutations and other mutations are constructed for each of the CDR grafts as follows.

For heavy chains VH5F9.1-GL, VH5F9.2-GL and VH5F9.3-GL, one or more of the following vernier and VH / VL connecting residues are backmutated as follows:
V37 → I, V48 → I, S49 → G and / or R98 → K.

For heavy chains VH5F9.4-GL, VH5F9.5-GL and VH5F9.6-GL, one or more of the following vernier and VH / VL connecting residues are backmutated as follows:
V37 → I, V48 → I, A49 → G, R98 → K.

For heavy chains VH5F9.7-GL, VH5F9.8-GL and VH5F9.9-GL, one or more of the following vernier and VH / VL connecting residues are backmutated as follows:
V37 → I, V48 → I, S49 → G.

Additional mutations include the following:
For heavy chain VH5F9.1-GL, VH5F9.2-GL and VH5F9.3-GL: D88 → A,
For heavy chains VH5F9.4-GL, VH5F9.5-GL and VH5F9.6-GL:
For Q1 → E and heavy chain VH5F9.7-GL, VH5F9.8-GL and VH5F9.9-GL:
L5 → V.

  For light chain VL 5F9.1-GL, one or more of the following vernier and VH / VL connecting residues are backmutated as follows: I2 → V, M4 → L, Y41 → F.

  For light chain VL 5F9.2-GL, one or more of the following vernier and VH / VL connecting residues are backmutated as follows: M4 → L, R51 → L.

  For light chain VL 5F9.3-GL, one or more of the following vernier and VH / VL connecting residues are backmutated as follows: M4 → L, Y41 → F.

(Example 7)
Construction and Expression of Recombinant Humanized Anti-RGM A Antibody pHybE expression with heavy and light chains containing framework back mutations to temporarily produce full-length humanized antibodies as described in section ix above The vector was co-transfected into 293-6E cells. Mutations were introduced into the CDR grafted antibody sequences as prepared according to Example 5 by de novo synthesis of variable domains and / or using mutagenic primers and PCR and methods well known in the art. The amino acid sequences of the VH and VL regions of the humanized antibody are disclosed in Table 8.

Specifically for heavy chains:
VH5F9.1, VH5F9.5 and VH5F9.9 contain VH5F9.4-GL with a Q1 → E mutation.

  VH5F9.2, VH5F9.6, VH5F9.10, VH5F9.19, VH5F9.20, VH5F9.21, and VH5F9.22 are the Q1 → E mutation and the next vernier and VH / VL connecting residue back mutation: V37 Contains VH5F9.4-GL with → I, V48 → I, A49 → G, and T98 → K.

  VH5F9.3, VH5F9.7 and VH5F9.11 contain VH5F9.7-GL with the L5 → V mutation. VH5F9.4, VH5F9.8, VH5F9.12, VH5F9.23, VH5F9.24, VH5F9.25 and VH5F9.26 are the L5 → V mutation and the next vernier and VH / VL connecting residue backmutation: V37 → I, V48 → I, S49 → G contains VH5F9.7-GL.

For the light chain:
VL5F9.1, VL5F9.2, VL5F9.3 and VL5F9.4 are the same as VL5F9.1-GL.

  VL5F9.5, VL5F9.6, VL5F9.7 and VL5F9.8 are identical to VL5F9.2-GL.

  VL5F9.9, VL5F9.10, VL5F9.11 and VL5F9.12 are the same as VL5F9.3-GL.

  VL5F9.19 and VL5F9.23 contain VL5F9.2-GL with the following vernier and VH / VL connecting residue backmutations: M4 → L, R51 → L. VL5F9.20 and VL5F9.24 contain VL5F9.2-GL with the following vernier and VH / VL connecting residue backmutations: M4 → L.

  VL5F9.21 and VL5F9.25 contain VL5F9.3-GL with the following vernier and VH / VL connecting residue backmutations: M4 → L, Y41 → F. VL5F9.22 and VL5F9.26 contain VL5F9.3-GL with the following vernier and VH / VL connecting residue backmutations: M4 → L.

(Example 8)
Characterization of humanized 5F9 antibody using competitive ELISA ELISA plates (50 μL / well of 0.25 μg / mL hRGM A in 0.2 M carbonate-sodium bicarbonate buffer, pH 9.4 overnight at 4 ° C. Costar 3369), washed with wash buffer (PBS containing 0.1% Tween 20), and blocked with 200 μL / well of 2% nonfat dry milk in PBS for 1 hour at room temperature. Mixture of biotinylated chimera 5F9 (0.1 μg / mL final concentration) and unlabeled competitive test antibody serially diluted 5-fold starting from 50 μg / mL final concentration in 50 μL / well of ELISA buffer after washing with wash buffer Was added in duplicate. Plates were incubated for 1 hour at room temperature, washed with wash buffer, and bound antibody was detected using 100 μL / well of a 1: 10,000 dilution of HRP-conjugated streptavidin (Fitzgerald) in ELISA buffer did. After incubating at room temperature for 1 hour and washing with a washing buffer, color was developed by adding 100 μL / well of TMB buffer (Zymed). After incubation at room temperature for 15 minutes, color development was stopped by adding 50 μL / well of 1N hydrochloric acid. Absorbance was read at 490 nm.

Table 9 shows the IC ₅₀ values of humanized 5F9 antibody obtained using the computer software GraphPad Prism (GraphPad Software Inc., San Diego, Calif.).

Example 9
Affinity determination of chimeric and humanized antibodies using BIACORE technology The BIACORE assay (Biacore, Inc, Piscataway, NJ) determines antibody affinity by kinetic measurements of association and dissociation rate constants. Continuous HBS-EP at 25 ℃ (10mM HEPES [pH7.4] , 150mM NaCl, 3mM EDTA and 0.005% Surfactant P20) was ^{used, Biacore} (R) 3000 instrument ^(Biacore (R) AB Antibody binding to recombinant purified human RGM A was determined by measurements based on surface plasmon resonance at Uppsala, Sweden). All chemicals ^were obtained from Biacore (R) AB (Uppsala, Sweden). Fragment-specific polyclonal diluted in goat anti-human IgG approximately 5000 RU, (Fcγ), 10 mM sodium acetate (pH 4.5) using a standard amine coupling kit according to the manufacturer's instructions and procedures at 25 μg / mL The antibody (Pierce Biotechnology Inc, Rockford, IL) was immobilized directly across the CM5 research grade biosensor chip. Unreacted parts on the biosensor surface were blocked with ethanolamine. The modified carboxymethyl dextran surface of flow cells 2 and 4 was used as the reaction surface. Unmodified carboxymethyl dextran without goat anti-human IgG from flow cells 1 and 3 was used as the reference surface. The purified antibody was diluted in HEPES-buffered salt for capture across the goat anti-human IgG specific reaction surface. A human antibody to be captured as a ligand (25 μg / mL) was injected over the reaction platform at a flow rate of 5 μL / min. The association and dissociation rate constants, k _on (unit M ⁻¹ s ⁻¹ ) and k _off (unit s ⁻¹ ) were examined under a continuous flow rate of 25 μL / min. Rate constants were obtained by performing kinetic binding measurements at 10 different antigen concentrations ranging from 0.39 to 50 nM. For kinetic analysis, using the Biavaluation 4.0 software, the kinetic equations obtained from the 1: 1 Langmuir binding model were similarly applied to the association and dissociation phases of all 8 injections (using global fit analysis). And fit). Next, the equilibrium dissociation constant (unit M) of the reaction between the humanized antibody and recombinant purified human RGM A was calculated from the reaction rate constant by the following formula: K _D = k _off / k _on .

(Example 10)
The humanized 5F9 antibody neutralizes the chemical repulsive activity of human RGM A in the neuronal SH-SY5Y chemotaxis assay.

Chemotaxis assays measure the chemotaxis behavior of cells in response to diffusible factors that can exert chemoattractant or chemical repulsion activity. RGM A has been described as a protein that acts in both membrane-bound (contact-dependent repulsion) and soluble diffusive forms (chemical repulsion) and has therefore been evaluated in the hRGM A chemotaxis assay. For this purpose, RGM A-sensitive human neuroblastoma cells SH-SY5Y carrying the RGM receptor neogenin were used (Schaffar et al., J. Neurochemistry: 107: 418-431, 2008). SH-SY5Y cells were grown in Earl Balanced Salt Solution / F12 (EBSS / F12) medium supplemented with 10% fetal bovine serum and 1% non-essential amino acids (MEM-NEAA). For induction of neurite outgrowth, cells were cultured in medium supplemented with 10 μm retinoic acid (RA). After 5-6 hours, the cells were trypsinized and counted for entry into a 24-well Boyden Chambers (BD Falcon 351185, HTS Multiwell System). 500 μL of cell suspension (corresponding to 1 × 10 ⁵ cells) was added to the inner circle of each well. This inner circular portion is separated from the larger outer circular portion of each well by a PET film having an 8 μm pore diameter. 600 μL of medium +/− RGM A +/− antibody was pipetted into the outer circle and the cells were cultured overnight at 37 ° C. in a multiwell Boyden chamber. After incubation, the medium was removed by aspiration and replaced with fixative (2% paraformaldehyde). Fixation was continued for 2 hours at room temperature, and after several washing steps with PBS, permeabilization was performed using PBS containing 0.1% Triton-X-100 (15 minutes, RT). Cells were stained by incubating them for 1 hour in the dark in a solution of Alexa Fluor 488 phalloidin 1: 100 (Invitrogen A12379) and bisbenzimide (H332456) 1: 100. After two washing steps with PBS, the cultures were filled with PBS, sealed with parafilm, and stored in the dark for analysis under a fluorescence microscope (Zeiss Axiovert).

  In the absence of hRGM A, cells migrate through the membrane pores and can be counted after fixation and staining. Only the cells that adhere to the bottom of the membrane are counted because these cells have migrated through the PET membrane. Prior to the fixation procedure, the cells on the top side of the membrane were carefully detached. This chemotaxis assay demonstrated that in the presence of hRGM A, the number of SH-SY5Y cells migrating through the membrane was significantly reduced by more than 80%. Rat monoclonal 5F9, chimeric human-rat 5F9 and humanized 5F9 partially or fully neutralize the chemical repulsive activity of hRGM A at 10 μg / mL, as evidenced by the increased number of cells seen at the bottom of the membrane. (The isotype control rat monoclonal antibody (p21) was not neutralized) (FIG. 10).

(Example 11)
5F9 induces regeneration of destroyed damaged optic nerve axons in a rat model of optic nerve injury.

  The optic nerve destruction (or optic nerve injury) model provides an animal model for testing various substances that stimulate optic nerve fiber regeneration and reduce massive cell death of retinal ganglion cells.

  This experiment was performed in adult male Sprague Dawley and male Wistar rats obtained from Charles River (D) Laboratories (Germany). These animals were maintained in one cage with a 12:12 hour light / dark cycle with constant feeding. The optic nerve destruction is always performed only in the left eye with minimal anterior surgery. This is a minimally invasive method of optic nerve injury and was developed by the inventors according to the human visual surgical method. Prior to and during the surgical procedure, the animals are anesthetized by inhalation anesthesia using sevoflurane (Abbott GmbH Co. & KG, Delkenheim, Germany) and secured to the operating table using a jaw clamp and adhesive tape for the limbs. Prevent the hypothermia by placing the animal on a heating pad. The left eye is carefully removed from the ligament and connective tissue for anterior destruction of the rat optic nerve. As a first step, the adjacent tissue at the outer corner of the eye is cut under a microscope (2-3 mm). The optic nerve is then exposed by using forceps to bring the eye muscles and lacrimal gland to the side (and thus save it). In the next step, the meninges were opened longitudinally by using a microfistula to expose the optic nerve.

  As a result, the mobility of the eye is enhanced, the external rotation of the eye is possible, and the left optic nerve can be touched. Damage is applied to the optic nerve approximately 1-3 mm after the eye using forceps to provide a constant maximum pressure over 20-30 seconds. Special care is taken not to damage the vascular supply to the eye.

Topical administration of antibody and buffer solution After injury to the optic nerve, male Sprague Dawley rats were topically treated with 5F9 antibody (n = 10), 8D1 control antibody (n = 10) or with vehicle control PBS (n = 10). Was treated. The experiment was performed blind to different treatment groups. For local antibody administration, a small gel foam piece (length: 2.5 mm, width: 2.5 mm, height: 2.5 mm) is soaked in 20 μL of 10 mg / mL antibody solution or 20 μL of PBS and immediately adjacent to the optic nerve lesion site. Put it on. After minimally invasive surgery and antibody application, the animals were placed on a paper towel in a clean cage placed on a warming device to regulate body temperature until the animals began to move. An ointment containing antibiotics (Gentamyltrex, Dr. Mann Pharma) was applied to the eye to prevent bacterial infection and scleral dryness. Immediately after surgery and then twice daily for 3 days, carprofen (Rimadyl, 5 mg / kg, Pfizer GmbH, Karlsruhe) was administered i. p. Administered. To confirm that all animals survived and recovered from anesthesia and surgery, the animals were observed and adjusted regularly for several hours immediately after surgery and the next day. Five weeks after surgery and antibody / vehicle application, animals were anesthetized with an overdose of narcolene (40-60 mg / kg) and perfused by infusion of 4% paraformaldehyde solution into the heart. The optic nerve was removed and transferred to a 4% paraformaldehyde solution for 1 hour at room temperature to ensure proper fixation of the tissue. After postfixation, the rat optic nerve was stored overnight in a 30% sucrose solution (4 ° C.). The next day, the optic nerve was embedded in Tissue Tek, frozen, and a section of a longitudinal section having a thickness of 16 μm was prepared using a cryostat.

  For immunostaining, optic nerve sections were fixed with cold (−20 ° C.) acetone (10 minutes), washed 3 × with Tris-buffered saline (TBS, Fluka 93312) (5 minutes), blocked, and treated with 5% bovine. Permeabilization was performed at room temperature with TBS containing serum albumin and 1% Triton-X-100 (30 minutes). Residual BSA and surfactant were removed by two separate washing steps (5 minutes) with TBS. Sections were incubated for 1 hour at room temperature with polyclonal rabbit anti-GAP-43 antibody (Abcam, ab7562) diluted 1: 100 in 5% BSA / TBS solution. After 3 washing steps with TBS, 0.1% Tween, 1 in 5% BSA / TBS containing a 1: 100 dilution of bisbenzimide (H33258, 50 μg / mL) to visualize cell nuclei. Sections were incubated with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (Molecular Probes A11034) diluted 1000 for 1 hour at room temperature. Prior to embedding, the stained sections were washed 3 times with TBS 0.1% Tween (5 minutes for each step) and distilled water. Sections were embedded in Fluoromount G, covered with coverslips and stored in the dark for microscopic recording.

  Using Zeiss fluorescence microscope images, images of stained longitudinal sections (FIG. 11) were saved using Zeiss Axiovision software. A single image of each nerve was mounted for analysis using Photoshop Image Analysis software (Adobe). Using the synthesized image of the optic nerve, quantitative analysis was performed by two different methods. GAP-43 positive area of the lesion site was measured using Axiovision software. (FIG. 12B). Independent of this first quantitative analysis, single regenerated fibers (GAP-43 positive) were counted in four different areas: 0-200 μm, 200-400 μm, 400-600 μm and 600-1200 μm from the fracture site. It was. Data analysis and statistical evaluation of the data were performed using Graphpad Prism software (FIG. 12A).

Systemic administration of antibody and buffer solution For systemic antibody delivery, male Wistar rats were systemically (intraperitoneally (ip) or intravenously (5) with 5F9 antibody (n = 10 mice) or vehicle control PBS (n = 10 mice). Treated in iv)). The animals were injected twice and injected immediately after induction of nerve destruction, on day 0, and on day 21 after destruction. The dose of antibody given was 2 mg / kg on day 0 and 10 mg / kg on day 21. The animals were sacrificed 5 weeks after the destructive injury and subjected to tissue excision, sectioning, staining and quantitative analysis as described above. As before, the experiment was conducted blindly on two different treatment groups. A composite image of the rat optic nerve is shown in FIG. In 5F9 treated animals (A), in contrast to control animals treated with PBS (B), many GAP-43 positive fibers extend beyond the break site. The destruction site is at the left end and the regenerated fiber is stained with an antibody against GAP-43. Many fibers are observed at the upper and lower edges of the optic nerve of 5F9-treated animals, but not in PBS animals.

  5F9 significantly increased the number of regenerated GAP-43 positive fibers, but not the vehicle control PBS. In animals treated with 5F9, significantly more fiber (p <0.001) was found at a distance of 300 μm to 1800 μm than vehicle-treated animals. Animals were treated on days 0 and 21 with 5 F9 at 2 mg / kg and 10 mg / kg, respectively. Antibody or vehicle was administered intraperitoneally or intravenously. Data are from analysis of 9 animals per group. Three consecutive cryostat sections per animal were analyzed (FIG. 14A).

  In a second experiment, male Wistar rats were systemically (iv) following optic nerve injury with 5F9 antibody (n = 10), 8D1 control antibody (n = 10) or with vehicle control PBS (n = 10). Was treated. Rats were injected once a week with 2 mg / kg of antibody administered iv and started immediately after optic nerve destruction. All rats received 4 injections and the animals were euthanized 5 weeks after the destructive injury. Experiments were performed blindly and tissue processing and quantitative analysis were performed as previously described. 5F9 significantly increased the number of regenerated GAP-43 positive fibers, but not the vehicle control PBS. Significantly more fibers were seen in 5F9 treated animals than at vehicle or control antibody treated animals at distances from 200 μm to 1400 μm (p <0.001). Animals were iv treated weekly for 4 weeks starting on day 0 with 5F9 (2 mg / kg per dose), with control antibody 8D1 (2 mg / kg per dose) or with PBS (FIG. 14B).

(Example 11)
In a rat model of optic nerve injury, 5F9 is disrupted and induces remyelination of damaged optic nerve axons.

  A marker for oligodendrocytes and myelin is myelin basic protein (MBP). To answer the question of whether remyelination differs in different treatment groups, antibodies against MBP were used. To visualize the process of remyelination, optic nerve sections of systemically treated animals were fixed with cold (−20 ° C.) acetone (10 min) and 3 with Tris-buffered saline (TBS, Fluka 93312). Washed once (5 minutes), blocked, and permeabilized with TBS containing 5% bovine serum albumin and 1% Triton-X-100 at room temperature (30 minutes). Residual BSA and surfactant were removed by two separate washing steps with TBS (5 minutes each). Sections were incubated at 4 ° C. for 3 hours or overnight with a polyclonal rabbit anti-MAB antibody (Abcam, ab2404) diluted 1:50 in 5% BSA / TBS solution. After 3 washing steps with TBS, 0.1% Tween, 1 in 5% BSA / TBS containing a 1: 100 dilution of bisbenzimide (H33258, 50 μg / mL) to visualize cell nuclei. Sections were incubated with Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (Molecular Probes A11034) diluted 1000 for 1 hour at room temperature. Prior to embedding, the stained sections were washed 3 times with TBS 0.1% Tween (5 minutes for each step) and with distilled water. Sections were embedded in Fluoromount G, covered with a coverslip and stored in the dark for microscopic recording.

  Images of stained longitudinal sections were stored using Zeiss Axiovision software using a Zeiss fluorescence microscope. A single image of each nerve was mounted for analysis using Photoshop Image Analysis software (Adobe). Using the synthesized image of the optic nerve, quantitative analysis was performed by two different methods. The MBP positive area of the lesion site was measured using Axiovision software. Data analysis and statistical evaluation of the data was performed using Graphpad Prism software.

  Animals were treated on days 0 and 21 with 5 F9 at 2 mg / kg and 10 mg / kg, respectively. Antibody or vehicle was administered intraperitoneally or intravenously.

Synthetic image of rat optic nerve Myelin formation is visualized using an antibody against myelin marker, myelin basic protein MBP. The site of destruction is located in the middle of the composite nerve, which is not present in vehicle-treated control animals (A and B). In 5F9-treated animals (C and D), a number of MBP-positive structures are observed in the central region of the optic nerve (mid-destruction) (FIG. 15).

  Myelin formation is visualized using an antibody against the myelin marker myelin basic protein MBP. MBP area was measured using Zeiss Axiovision software. M1 and M2 are two independent measurements, and M is the average measured MPB positive area. 5F9 significantly increases the MBP area at the site of optic nerve destruction (p <0.001 versus vehicle control) by a factor of 3.5 (FIG. 16).

Claims (91)

Dissociates from human RGM A with 1x10 ^-7 M following _{K D} and ^1x10 -2 ^{s -1} following _{k off} rate constant (both determined by surface plasmon resonance), the binding protein.   A binding protein that binds to human RGM A and neutralizes neurite outgrowth inhibitory activity of human RGM A as measured in a standard in vitro assay. The following additional functional characteristics:
Binding to rat RGM A,
3. The binding protein of one of claims 1 and 2, having at least one of binding to human RGM C binding to rat RGM C.   4. The binding protein of one of claims 1 to 3, which modulates the ability of RGM to bind to at least one RGM receptor.   5. The binding protein of claim 4 that interacts with the receptor binding domain of human RGM A. The following interaction:
5. The binding protein of claim 4 that modulates at least one of binding of human RGM A to human BMP-4 binding of hRGM A to human neogenin binding of hRGM C to human neogenin.   The binding protein according to one of claims 1 to 6, which is a humanized antibody. 8. The binding protein according to one of claims 1 to 7, comprising an antigen binding domain (the binding protein is capable of binding to an epitope of an RGM molecule, wherein the antigen binding domain is
GTTPDY (SEQ ID NO: 59),
FQATHDPLT (SEQ ID NO: 62),
ARRNEYYGSSSFFDY (SEQ ID NO: 65),
LQGYIPPRT (SEQ ID NO: 68) and at least one CDR comprising an amino acid sequence selected from the group consisting of a modified CDR amino acid sequence having at least 50% sequence identity with one of said sequences. ). A binding protein comprising an antigen binding domain (the binding protein is capable of binding to an epitope of an RGM molecule, the antigen binding domain comprising:
GTTPDY (SEQ ID NO: 59),
FQATHDPLT (SEQ ID NO: 62),
ARRNEYYGSSSFFDY (SEQ ID NO: 65),
LQGYIPPRT (SEQ ID NO: 68) and at least one CDR comprising an amino acid sequence selected from the group consisting of a modified CDR amino acid sequence having at least 50% sequence identity with one of said sequences. ).   At least one amino acid sequence selected from the group consisting of SEQ ID NOs: 57, 58, 60, 61, 63, 64, 66, 67 and a modified CDR amino acid sequence having at least 50% sequence identity with one of said sequences 10. The binding protein according to one of claims 1 to 9, further comprising a CDR. The at least one CDR is

The binding protein according to any one of claims 1 to 10, comprising an amino acid sequence selected from the group consisting of:

Or at least one of the three CDRs comprises at least three CDRs selected from the variable domain set that is a modified CDR amino acid sequence having at least 50% sequence identity with the parent sequence. 11. The binding protein according to 11.   13. The binding protein of claim 12, comprising at least two variable domain CDR sets. The at least two variable domain CDR sets are:
14. A binding protein according to claim 13 selected from the group consisting of VH5F9 set and VL5F9 set and VH8D1 set and VL8D1 set.   The binding protein according to one of claims 1 to 14, further comprising a human acceptor framework.   The human acceptor framework is a group consisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 and 33 16. A binding protein according to claim 15 comprising at least one amino acid sequence selected from. VH3-48 set (SEQ ID NOS: 15, 16, and 17)
VH3-33 set (SEQ ID NOS: 21, 22, and 23)
VH3-23 set (SEQ ID NO: 24, 25 and 26)
(Each of these sets
JH3 (SEQ ID NO: 18),
JH4 (SEQ ID NO: 19),
JH6 (SEQ ID NO: 20)
Combined with an additional framework sequence selected from Or A18 set: (SEQ ID NO: 27, 28 and 29)
A17 set: (SEQ ID NO: 31, 32 and 33)
(Each of these sets is selected from the group consisting of a set of additional framework sequences selected from JK2 (SEQ ID NO: 2).)
17. A binding protein according to claim 16 comprising a set of framework sequences.   At least one heavy chain variable domain selected from SEQ ID NOs: 35, 36, 37, 38, 39, 40, 41, 42 and 43; and / or at least one light chain selected from SEQ ID NOs: 44, 45 and 46 The binding protein according to any one of claims 1 to 17, comprising a variable domain. Two variable domains, wherein the two variable domains are
SEQ ID NOs: 35 and 44; 36 and 44; 37 and 44; 38 and 44; 39 and 44; 40 and 44; 41 and 44; 42 and 44;
SEQ ID NOs: 35 and 45; 36 and 45; 37 and 45; 38 and 45; 39 and 45; 40 and 45; 41 and 45; 42 and 45;
SEQ ID NOs: 35 and 46; 36 and 46; 37 and 46; 38 and 46; 39 and 46; 40 and 46; 41 and 46; 42 and 46;
19. The binding protein of claim 18 having an amino acid sequence selected from. The human acceptor framework comprises at least one framework region amino acid substitution at a key residue, wherein the key residue is
Residues adjacent to the CDRs;
Glycosylation site residues;
Rare residue;
Residues capable of interacting with the RGM epitope;
Residues capable of interacting with CDRs;
Canonical residues;
Contact residues between the heavy and light chain variable regions;
Residues in the vernier zone;
An N-terminal residue capable of forming paraglutamate; and a residue in the region overlapping between the variable heavy chain CDR1 defined by Chothia and the first heavy chain framework defined by Kabat 20. A binding protein according to any one of claims 15 to 19 selected from the group. The key residue is
(Heavy chain sequence position): 1, 5, 37, 48, 49, 88, 98
(Light chain sequence position): 2, 4, 41, 51
21. The binding protein of claim 20, wherein the binding protein is selected from the group consisting of:   The binding protein according to any one of claims 1 to 21, wherein the binding protein is a consensus human variable domain.   The human acceptor framework comprises at least one framework region amino acid substitution, the amino acid sequence of the framework is at least 65% identical to the sequence of the human acceptor framework, and the human acceptor framework 23. A binding protein according to any one of claims 15 to 22 comprising at least 70 amino acid residues which are identical. SEQ ID NOs: 47, 48, 49, 50;
SEQ ID NOs: 51, 52, 53 and 54
24. The binding protein of any one of claims 1 to 23, comprising at least one (mutated framework) variable domain having an amino acid sequence selected from the group consisting of: Two variable domains, wherein the two variable domains are
SEQ ID NOs: 47 and 44; 47 and 45; 47 and 46; 47 and 51; 47 and 52; 47 and 53; 47 and 54;
48 and 44; 48 and 46; 48 and 51; 48 and 52; 48 and 53; 48 and 54;
49 and 44; 49 and 46; 49 and 51; 49 and 52; 49 and 53; 49 and 54;
SEQ ID NOs: 50 and 44; 50 and 45; 50 and 46; 50 and 51; 50 and 52; 50 and 53; 50 and 54
25. The binding protein of claim 24, having an amino acid sequence selected from the group consisting of:   26. A binding protein according to any one of claims 1 to 25 capable of binding to a target selected from RGM molecules.   27. The binding protein of any one of claims 1 to 26, capable of binding to human RGM A. The following additional functional characteristics:
Binding to rat RGM A,
Binding to human RGM C,
28. The binding protein of claim 27, having at least one of binding to rat RGM C.   29. A binding protein according to any one of claims 1 to 28, capable of modulating the biological function of a target selected from RGM molecules.   30. The binding protein of claim 29, wherein said binding protein modulates the ability of RGM to bind to at least one receptor of RGM. The following interaction:
Binding of human RGM A to human BMP-4,
Binding of hRGM A to human neogenin,
Binding of hRGM C to human neogenin,
32. The binding protein of claim 30, which modulates at least one of the binding of human RGM A to human BMP-2.   32. A binding protein according to any one of claims 1 to 31 capable of inhibiting RGM biological activity.   35. The binding protein of claim 32, wherein the RGM molecule is RGM A.   34. The binding protein of claim 33, wherein RGM A is selected from human, cynomolgus monkey, rat, chick, frog and fish. At least about ¹⁰ 2 ^{M -1 s} -1; at least about ¹⁰ 3 ^{M -1 s} -1; at least about ¹⁰ 4 ^{M -1 s} -1; at least about ¹⁰ 5 ^{M -1 s} -1; at least about ¹⁰ 6 M ^- Claim 1 to Claim 1 having an association rate constant (k _on ) for said target selected from the group consisting of ¹ s ^-1 and at least about 10 ⁷ M ^-1 s ^-1 (as measured by surface plasmon resonance). 35. The binding protein of any one of items 34. Up to about 10 ⁻² s ⁻¹ ; up to about 10 ⁻³ s ⁻¹ ; up to about 10 ⁻⁴ s ⁻¹ ; up to about 10 ⁻⁵ s ⁻¹ ; and up to about 10 ⁻⁶ s ⁻¹ (by surface plasmon resonance). 36. The binding protein of any one of claims 1 to 35, having a dissociation rate constant ( _koff ) for said target selected from the group consisting of: Up to about 10 ⁻⁷ M; up to about 10 ⁻⁸ M; up to about 10 ⁻⁹ M; up to about 10 ⁻¹⁰ M; up to about 10 ⁻¹¹ M; up to about 10 ⁻¹² M; and up to 10 ⁻¹³ M 37. A binding protein according to any one of claims 1 to 36 having a dissociation constant (K _D ) for said target selected from the group.   38. An antibody construct comprising a binding protein according to any one of claims 1 to 37, further comprising a linker polypeptide or an immunoglobulin constant domain. The binding protein is
Immunoglobulin molecules,
Monoclonal antibodies,
Chimeric antibody,
CDR grafted antibody,
Humanized antibodies,
Fab,
Fab ',
F (ab ′) 2,
Fv,
Disulfide bond Fv,
scFv,
Single domain antibody,
Diabody,
Multispecific antibodies,
Bispecific antibodies,
40. The antibody construct of claim 38, wherein the antibody construct is selected from the group consisting of a dual variable domain immunoglobulin and a bispecific antibody. The binding protein is
A human IgM constant domain,
Human IgG1 constant domain,
Human IgG2 constant domain,
Human IgG3 constant domain,
Human IgG4 constant domain,
Human IgE constant domain,
A human IgD constant domain,
Human IgA1 constant domain human IgA2 constant domain,
40. The antibody construct of any one of claims 38 and 39, comprising a heavy chain immunoglobulin constant domain selected from the group consisting of a human IgY constant domain and a corresponding mutated domain.   41. The antibody construct according to any one of claims 38 to 40, comprising an immunoglobulin constant domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 12, 13, and 14.   42. An antibody complex comprising an antibody construct according to any one of claims 38 to 41, further comprising an agent selected from the group consisting of an immunoadhesion molecule, a contrast agent, a therapeutic agent and a cytotoxic agent.   43. The antibody complex according to claim 42, wherein the agent is a contrast agent selected from the group consisting of a radioactive label, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, and biotin. The contrast agent is a radioactive label selected from the group consisting of ³ H, ¹⁴ C, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹²⁵ I, ¹³¹ I, ¹⁷⁷ Lu, ¹⁶⁶ Ho, and ¹⁵³ Sm. Item 44. The antibody complex according to Item 43.   A therapeutic agent or cell selected from the group consisting of an antimetabolite, an alkylating agent, an antibiotic, a growth factor, a cytokine, an angiogenesis inhibitor, an antimitotic agent, an anthracycline, a toxin and an apoptotic agent. 44. The antibody conjugate according to claim 43, which is a toxic agent.   42. The antibody construct of any one of claims 38 to 41, wherein the binding protein retains a human glycosylation pattern.   46. The antibody conjugate of any one of claims 42 to 45, wherein the binding protein retains a human glycosylation pattern.   38. A binding protein according to any one of claims 1 to 37 present as a crystal.   42. The antibody construct according to any one of claims 38 to 41 present as a crystal.   46. The antibody complex according to any one of claims 42 to 45, wherein the antibody construct is present as a crystal.   49. The binding protein of claim 48, wherein the crystal is a carrier-free pharmaceutical controlled release crystal.   50. The antibody construct of claim 49, wherein the crystal is a carrier-free pharmaceutical controlled release crystal.   51. The antibody conjugate of claim 50, wherein the crystal is a carrier-free pharmaceutical controlled release crystal.   49. The binding protein of claim 48, which has a longer half-life in vivo than its soluble form counterpart.   50. The antibody construct of claim 49, which has a longer half-life in vivo than its soluble form counterpart.   51. The antibody conjugate of claim 50, which has a longer half-life in vivo than its soluble form counterpart.   49. The binding protein of claim 48, which retains biological activity.   50. The antibody construct of claim 49, which retains biological activity.   51. The antibody conjugate of claim 50 that retains biological activity.   38. An isolated nucleic acid encoding the binding protein amino acid sequence of any one of claims 1 to 37.   42. An isolated nucleic acid encoding the antibody construct amino acid sequence of any one of claims 38 to 41.   46. An isolated nucleic acid encoding the antibody conjugate amino acid sequence of any one of claims 42 to 45.   63. A vector comprising the isolated nucleic acid according to any one of claims 60 to 62.   64. The vector of claim 63, selected from the group consisting of pcDNA, pTT, pTT3, pEFBOS, pBV, pJV, pHybE and pBJ.   A host cell comprising the vector of any one of claims 63 and 64.   66. The host cell of claim 65, which is a prokaryotic cell.   E. 68. The host cell of claim 66, wherein the host cell is E. coli.   68. The host cell of claim 67, which is a eukaryotic cell.   69. The host cell according to claim 68, wherein the eukaryotic cell is selected from the group consisting of protist cells, animal cells, plant cells and fungal cells.   70. The host cell of claim 69, wherein the eukaryotic cell is an animal cell selected from the group consisting of a mammalian cell, an avian cell and an insect cell.   70. The host cell according to claim 69, selected from HEK cells, CHO cells, COS cells and yeast cells.   72. The host cell of claim 71, wherein the yeast cell is Saccharomyces cerevisiae.   71. A host cell according to claim 70 which is an insect Sf9 cell.   74. Binding to RGM comprising culturing the host cell of any one of claims 65 to 73 in a medium under conditions sufficient to produce a binding protein capable of binding to RGM. To produce a protein that can be produced.   75. A protein produced according to the method of claim 74. (A) a formulation (the formulation comprises the crystallization product protein of any one of claims 48 to 50) and ingredients;
(B) at least one polymeric carrier;
A composition for the release of bound protein.   The polymeric carrier is poly (acrylic acid), poly (cyanoacrylate), poly (amino acid), poly (anhydride), poly (depsipeptide), poly (ester), poly (lactic acid), poly (lactic acid-glycol) Acid) copolymer or PLGA, poly (b-hydroxybutyrate), poly (caprolactone), poly (dioxanone); poly (ethylene glycol), poly ((hydroxypropyl) methacrylamide, poly [(organo) phosphazene], poly ( Ortho ester), poly (vinyl alcohol), poly (vinyl pyrrolidone), maleic anhydride-alkyl vinyl ether copolymer, pluronic polyol, albumin, alginate, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharide, glucose 77. The composition of claim 76, wherein the composition is a polymer selected from one or more of the group consisting of licaminoglycans, sulfated polysaccharides, mixtures thereof and copolymers.   77. The composition of claim 76, wherein the component is selected from the group consisting of albumin, sucrose, trehalose, lactitol, gelatin, hydroxypropyl- [beta] -cyclodextrin, methoxypolyethylene glycol and polyethylene glycol.   79. A method for treating a mammal comprising administering to the mammal an effective amount of a composition according to any one of claims 77 and 78.   60. A pharmaceutical composition comprising the product of any one of claims 1 to 59 and a pharmaceutically acceptable carrier.   81. The pharmaceutical composition of claim 80, wherein the pharmaceutically acceptable carrier functions as an adjuvant useful for improving absorption or dispersion of the binding protein.   82. The pharmaceutical composition of claim 81, wherein the adjuvant is hyaluronidase.   83. The pharmaceutical composition of claim 82, further comprising at least one additional therapeutic agent for treating a disease in which RGM activity is detrimental.   Said additional agents are therapeutic agents, contrast agents, cytotoxic agents, angiogenesis inhibitors; kinase inhibitors; costimulatory molecule blockers; adhesion molecule blockers; anti-cytokine antibodies or functional fragments thereof; methotrexate; cyclosporine; rapamycin; FK506; Possible label or reporter; TNF antagonist; anti-rheumatic agent; muscle relaxant, anesthetic, nonsteroidal anti-inflammatory drug (NSAID), analgesic, anesthetic, sedative, local anesthetic, neuromuscular blocker, antibacterial, psoriasis treatment Drugs, corticosteroids, anabolic steroids, erythropoietin, immunization, immunoglobulins, immunosuppressants, growth hormones, hormone supplements, radiopharmaceuticals, antidepressants, antipsychotics, stimulants, asthma drugs, beta agonists, inhalation Steroids, epinephrine or similar substances, cytokines and It is selected from the group consisting of a cytokine antagonist, a pharmaceutical composition of claim 83.   60. A method for reducing human RGM A activity comprising contacting human RGM A with a product of any one of claims 1 to 59 such that human RGM A activity is reduced.   60. A hRGM to a neogenin receptor in a subject comprising administering to a subject in need of decreasing hRGM A binding to a neogenin receptor the product of any one of claims 1 to 59. A method for reducing A binding.   60. Production according to any one of claims 1 to 59 for a subject in need of decreasing hRGM A binding to bone morphogenetic protein-2 and bone morphogenetic protein-4 (BMP-2 and BMP-4). A method for reducing hRGM A binding to bone morphogenetic protein-2 and bone morphogenetic protein-4 (BMP-2 and BMP-4) in the subject comprising administering an agent.   60. A method of treating a subject for a disease associated with RGM A activity comprising administering the product of any one of claims 1 to 59, alone or in combination with other therapeutic agents.   Administering the product of any one of claims 1 to 59, alone or in combination with other therapeutic agents, to a subject suffering from a disease in which RGM A activity is detrimental, A method for reducing RGM A activity in a subject.   Diseases are amyotrophic lateral sclerosis, brachial plexus injury, brain injury (including traumatic brain injury), cerebral palsy, Guillain Valley, cerebral white matter atrophy, multiple sclerosis, post-polio syndrome, spina bifida Spinal cord injury, spinal muscular atrophy, spinal tumor, stroke, transverse myelitis; dementia, senile dementia, mild cognitive impairment, Alzheimer-related dementia, Huntington's chorea, delayed dyskinesia, hyperactivity, Parkinson's disease, Steel-Richard syndrome, Down syndrome, myasthenia gravis, neurotrauma, vascular amyloidosis, cerebral hemorrhage with amyloidosis, encephalitis, acute confusion disorder, amyotrophic lateral sclerosis, glaucoma and Alzheimer 90. The method of claim 89, comprising a neurological disorder selected from the group comprising a disease.   52. An isolated CDR of a binding protein as defined in any one of claims 1 to 51.

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